In view of the finding that NfH aggregates in plasma might contribute to the ‘hook shaped curve’ shown in Fig. 2.2, I next developed a method to overcome this effect. Using plasma samples from 120-day-old SOD1G93A mice and age-matched WT controls, a number of established methods for the gentle disruption of NfH aggregates were therefore tested, including addition of a calcium chelator, such as EGTA or EDTA (Yabe et al., 2001a; Yabe et al., 2001b) or the pre-thawing of samples at 4°C (Petzold et al., 2003). As the results presented in Fig. 2.4 show, both of these methods improved the quantification of NfH levels during serial dilution. However, the error remained high, averaging 36% for pre-thawing (Fig. 2.4A), 63% for EDTA and 33% for EGTA (Fig. 2.4B). The effects of calcium chelators and/or prethawing were therefore not consistent enough to achieve parallelism.
As shown in Fig. 2.5, the most effective method for the disruption of NfH aggregates was found to be incubation in urea (Fig. 2.5). Urea incubation had a dose-dependent effect (0.5 M - 4 M) with the highest NfHSMI34 and NfHSMI35 levels detected following incubation of the sample diluent in 0.5 M urea at RT, 1 hour prior to incubation in the ELISA. Longer incubation periods (up to 24 hours at 4ºC) reduced the levels of NfH phosphoforms detected, with a decrease of up to 75% for NfHSMI34 and a 60% decrease for NfHSMI35 (Fig. 2.6).
Incubation of the plasma samples in 0.5 M urea-Barb2EDTA buffer at a 1:8 dilution for 1 hour at RT before adding to the ELISA abolished the hook shaped dilution curve and substantially improved parallelism (Fig. 2.7A & 2.7B).
A summary of the final optimised method for the detection of NfH phosphoforms in plasma is shown in Table 2.3. The Table details the protocol which overcomes the lack of parallelism and is therefore suitable for detection of NfH phosphoforms in i) plasma, and ii) small sample volumes.
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Figure 2.4
Figure 2.4. The effect of pre-thawing and calcium chelators on disruption of NfH aggregates. Both pre-thawing plasma samples (A) and addition of calcium
chelators (B) reduced the NfH “Hook Effect” observed during doubling dilutions, resulting in an averaged elevation of NfH levels during serial dilution of 36%, 63% and 33% for pre-thawing, EDTA and EGTA, respectively. Error bars: ±SEM.
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Figure 2.5
Figure 2.5. The effects of urea at different concentration on NfH levels in plasma of SOD1G93A mice. Plasma levels of both NfHSMI34 and NfHSMI35 largely increased after 1 hour urea-Barb2EDTA buffer incubation at room temperature, up to 47% at 0.5 M urea. Error bars: ±SEM.
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Figure 2.6
Figure 2.6. The effects of incubation period in Barb2EDTA buffer with urea at
various concentration on NfH levels in plasma of SOD1G93A mice. Levels of
NfHSMI34 and NfHSMI35 largely attenuated after 24-hour incubation at 4°C up to 75% and 60%, respectively.
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Figure 2.7
Figure 2.7. The effects of 1 hour incubation of 0.5 M Urea-Barb2EDTA buffer at
room temperature on plasma NfH levels in SOD1G93A mice. (A) Using serial
dilution of samples, more consistent NfH levels were detected at the 2nd (16%) and 3rd (11%) dilution. (B) In addition, the NfH ‘Hook Effect’ disappeared and parallelism was improved using the new urea-timed method (Incubation in 0.5 M Urea- Barb2EDTA buffer at room temperature for 1 hour). Error bars: ±S.E.M.
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Table 2.3 The optimised protocol for an ELISA detecting NfH in the plasma. Capture antibody Load 100 µl of Capture antibody (Covance SMI-34R or
SMI-35R in carbonate buffer1; v/w, 2/10000) into a 96-well microtitre plate*.
Incubate the plate at 4°C overnight.
Block the plate Decant the Capture antibody.
Rinse the plate twice with 150 µl wash solution3.
Block the plate with 100 µl blocking solution4 at RT for 1 hour on the shaker.
Sample preparation Add 5 µl of original plasma into 35 µl of Barb2EDTA buffer2 containing 0.5 M urea.
Well-mixed and incubate at RT for 1 hour----diluted plasma.
Load the samples Decant the blocking solution.
Rinse the plate twice with 150ul wash solution.
Load 95 µl of sample diluent5 into each well of the plate. Apply 5 µl of NfH standard (ranging from 0-100 ng/mL), the diluted and pre-incubated plasma, and the quality control sample in duplicates.
Incubate at RT for 1 hour on the shaker.
Detector antibody Decant the samples.
Wash the plate 3 times with 150 µl wash solution for 5 minutes on the shaker.
Load 100 µl of detector antibody (Sigma Rabbit anti-
neurofilament 200 in sample diluent; v/w, 10/10000) into the plate.
Incubate at RT for 1 hour on the shaker.
Reporter antibody Decant the detector antibody.
Wash the plate 3 times with 150 µl wash solution for 5 minutes on the shaker.
Load 100 µl of reporter antibody (DAKO Swine anti-rabbit HRP-linked antibody in sample diluent; v/w, 10/10000) into the plate.
Incubate at RT for 1 hour on the shaker.
Chemiluminescence readout
Decant the reporter antibody.
Wash the plate 6 times with wash solution for 5 minutes on the shaker.
Load 100 µl of TMB into the plate.
Incubate at RT for 20 minutes on the shaker in the dark. Stop the reaction with 50 µl of 1M HCL.
Read the plate at 450 nm, with 750 nm as the reference wavelength.
*All loading volume refers to volume adding into each well of the microtire plate.
1
Carbonate Buffer (pH 9.6): 13.85g anhydrose sodium carbonate and 26.10g sodium hydrogen carbonate in per litre distilled water
2
Barb2EDTA buffer (pH 8.6): 13.1g sodium barbitone, 2.1g barbitone, and 0.25g EDTA in per litre
distilled water
3
Wash solution: 0.1% bovine serum albumin (BSA) and 0.05% Tween 20 in Barb2EDTA buffer 4
Blocking solution: 1% BSA in Barb2EDTA buffer 5
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2.3.3 A comparison of plasma NfH levels in end-stage SOD1G93A mice with age-