6.3 TECHNIQUES FOR THE STUDY OF
6.3.2.2 Enzyme-Linked Immunosorbent Assay
Recently, our laboratory has begun to utilize ELISAs for the quantification of both pro- and antiinflammatory cytokines in neuropathic and radicular models of persistent pain. The detection of spinal cord cytokines is not limited by the available commercial ELISA kits since a laboratory in theory can make its own plates using existing available specific antibodies. However, we have found it more efficient to utilize commercial kits. As with the techniques to study mRNA, the first hurdle was to isolate protein from spinal cord tissue. For the present work, spinal cord homogenization
TABLE 6.2
Analysis of Commercial Sources of TNF, IL-1β, and IL-6 Antibodies for Reproducibility, Quality, and Specificity of Immunohistochemical Staining
Source (Catalogue Number)
Antibody
Description Dilution
Reproducible Staining
Staining Quality
(1:10) Pre- Absorbed
Antigen
(1:20) Pre- Absorbed
Antigen TNF
Endogen (PR-370)
PCa rabbit, anti-rat
1:1500 No Average 0d Not done
Genzyme Diagnostics (IP-400)
PC rabbit, anti-mouse
1:10,000 Yes Above
average
–f –
PeproTech Inc.
(500-P72)
PC rabbit, anti-rat
1:1000 Yes Below
average
+e Not done R&D Systems Inc.
(AF-510-NA)
PC goat, anti-rat
1:1500 No Below
average
– –
Santa Cruz Bio. Inc.
(Sc-1349)
PC goat, anti-rat
1:2500 Yes Average 0 –
Serotec Ltd.
(AAM12)
PC rabbit, anti-mouse/rat
1:3000 Yes Above
average
– –
IL-1β Endogen
(PR-427B)
PC rabbit, anti-rat
1:1500 No Average NC/SNRc Not done
Genzyme Diagnostics (LP-712)
PC rabbit, anti-human
1:1500 Yes Average 0 0
PeproTech Inc.
(500-P80)
PC rabbit, anti-rat
1:1000 No Average + Not done
R&D Systems Inc.
(AF-501-NA)
PC goat, anti-rat
1:1000 NC Average – +
Santa Cruz Bio. Inc.
(Sc-1252)
PC goat, anti-rat/mouse
1:2000 Yes Below
average
NC/SNR +
Serotec Ltd.
(MCA1397)
MCb mouse, anti-rat
1:1000 Yes Above
average
– Not done
IL-6 Endogen
(PR-627)
PC rabbit, anti-rat
1:1500 Yes Below
average
+ Not done
Genzyme Diagnostics (LP-716)
PC rabbit, anti-human
1:1000 Yes Above
average
– Not done
PeproTech Inc.
(400-06)
PC rabbit, anti-rat
1:1000 No Average NC/SNR Not done
Santa Cruz Bio. Inc.
(Sc-1266P)
PC goat, anti-rat/mouse
1:1500 Yes Below
average (perivas)
+ +
a PC = polyclonal antibody
bMC = monoclonal antibody
c NC/SNR = not conclusive/staining not reproducible
d0 is no change in staining quality or quantity following pre-absorption of antigen
e + is increased staining following pre-absorption of antigen
f – is decreased staining following pre-absorption of antigen
was adapted from the method of De La Monte et al.88 Once protein was isolated, we stored aliquots of protein at –80°C until the ELISA was run. Storage of samples in aliquots is ideal since repeated freeze–thaw cycles can degrade protein and thus degrade the detection of the target protein by ELISA.
A review of the ELISA literature revealed two ways to report data. Data have been reported as the weight of the protein of interest per either total tissue weight analyzed or per total protein in the sample. We completed a time course following L5 spinal nerve transection and examined IL-1β and IL-6 levels by both methods.
IL1-β protein concentrations were determined utilizing the quantitative sandwich enzyme immunoassay Quantikine® M rat IL-1β immunoassay (R&D Systems, Min- neapolis, MN) according to the manufacturer’s directions. IL-6 protein concentrations were determined utilizing Biosource IL-6 immunoassay (Biosource International, Camarillo, CA) according to the manufacturer’s directions. For the determination of total protein the BCA (bicinchoninic acid, Pierce Chemical Company, Rockford, IL) assay was utilized. We found that similar trends could be obtained by both methods of data presentation (Figs. 6.3 and 6.4) but significance was only achieved when IL-6 protein was calculated in terms of total tissue weight analyzed. Therefore, to detect FIGURE 6.3 Analysis of IL-6 protein by ELISA produced similar increases in protein at days 3 and 7 post-transection whether IL-6 protein was quantified in terms of àg of total protein (A) or in terms of tissue weight examined (B). IL-6 protein quantified per tissue weight revealed a statistically significant (*p < 0.05) increase in IL-6 in the L5 spinal cord on days 3 and 7 post-transection as compared to normal nạve animals (B). L5 spinal cord was isolated at 6 h (n = 4), 1 (n = 4), 3 (n = 8), or 7 (n = 7) d post L5 spinal nerve transection as well as from normal nạve animals (n = 6).
Normal 6 hours 1 day 3 days 7 days 100
80 60 40 20 0 pg IL-6/100 ug protein +/-SEM A
B
10 8 6 4 2 0
Normal 6 hours 1 day 3 days 7 days
+/-SEMng IL-6/g tissue
and characterize small changes in cytokines, we have chosen to report data in terms of tissue weight analyzed, which may preclude the detection of small changes in a specific cytokine due to the simultaneous upregulation of a large number of proteins at a rate disproportionate with the upregulation of the protein of interest.
The time-course data clearly exhibited increases in IL-6 protein levels at 3 and 7 d post L5 spinal nerve transection (Fig. 6.3), while IL-1β appeared upregulated on days 1 and 7 post-transection (Fig. 6.4). The temporal expression pattern of IL-1β and IL-6 protein following injury is reminiscent of the temporal expression patterns of mRNA as detected by RPA and thus, warrants the same implications of time points in determining whether a difference in the protein of interest will be observed or not. Unfortunately, analysis of central cytokine contributions to nociception is not as simple as just looking at the level of a specific cytokine in the lumbar spinal cord.
It must be remembered that there is endogenous regulation of cytokine expression and activity by concomitant expression of antiinflammatory cytokines, like IL-10 and IL-4, as well as endogenous cytokine antagonists. As already discussed in detail, regulation of cytokines at the message level also determines how much protein is synthesized. In addition, there are multiple points of regulation at the protein level, from the proteolytic cleavage of pro-proteins to the regulation of receptor and accessory protein expression. The complex regulation of the inflammatory cascade and the multiple proteins and signaling cascades involved highlights the challenge of studying the contribution of central cytokines to the etiology of neuropathic pain.
FIGURE 6.4 Analysis of IL-1β protein by ELISA produced similar trends whether IL-1β protein was quantified in terms of àg of total protein (A) or in terms of tissue weight (B) in L5 spinal cord tissue which was isolated at 6 h (n = 4), 1 (n = 4), 3 (n = 4), or 7 (n = 7) d post L5 spinal nerve transection as well as from normal nạve animals (n = 6).
Normal 6 hours 1 day 3 days 7 days 5
4 3 2 1 0 pg IL-1b/100 ug protein +/-SEM A
Normal 6 hours 1 day 3 days 7 days
+/-SEMng IL-1b/g tissue
B 0.5
0.4 0.3 0.2 0.1 0