Capítulo 1. Caracterización Territorial
1.2 Una mirada social de la UPZ
Standard curves All standard curves were run on the same plate to minimise the inter-assay variation. The concentration for the first antibody was 1:10000, for the second and third antibody 1:1000. The standard curves determined for the NfH capture antibodies were expressed as the percentage optical density (OD) for each calibrant.
This normalised procedure allows firstly direct comparison of the perfor- mance of the assay, which was e.g. linear for NfHSMI34(Figure 4.1) versus
quadratic for NfHSMI35(Figure 4.2). Secondly, those capturing antibod- ies which might still be worth using for the further development for an ELISA specific for one NfH phosphoform can be seen (e.g. SMI34 but not SMI311). The standard curves for the antibodies binding to phosphorylated NfH (NfHSMI34NfHSMI35NfHSMI310NfHNE14) are shown in Figures 4.1 to
4.4. The standard curves for the antibodies binding to non–phosphorylated NfH (NfHSMI32NfHSMI33NfHSMI37NfHSMI38NfHSMI311 ) are shown in Fig- ures 4.5 to 4.9.
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Figure 4.1: Standard curve for NfHSMI34. The regression line and 95%
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Figure 4.2: Standard curve for NfHSMI35. The regression line and 95%
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Figure 4.3: Standard curve for NfHSMI310. The regression line and 95%
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Figure 4.4: Standard curve for NfHNE14. The regression line and 95%
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Figure 4.5: Standard curve for NfHSMI32. The regression line and 95%
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Figure 4.6: Standard curve for NfHSMI33. The regression line and 95%
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Figure 4.7: Standard curve for NfHSMI37. The regression line and 95%
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Figure 4.8: Standard curve for NfHSMI38. The regression line and 95%
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Figure 4.9: Standard curve for NfHSMI311. The regression line and 95%
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Detection limit and analytical range The detection limit was deter- mined by the signal–to–noise ratio of the lowest calibrant to the blank (back- ground), Figure 4.10. An estimate for the analytical range for a particular antibody combination is the ratio of the highest calibrant to the noise (Fig- ure 4.11). The signal–to–noise ratio for SMI35 was about 4- to 10–fold and the analytical range 2- to 10–fold higher than for the other antibody com- binations. On the basis of these results SMI35 was selected as the capture antibody for the further development of the NfH ELISA.
Antibody concentration
NfH assay The first antibody was titrated against the second antibody. Dilutions used for the first antibody were 10001 , 50001 , 100001 and 1000001 . Di- lutions used for the second antibody were 1
500, 10001 and 50001 . All standard
curves were run in on the same plate. The results are summarised in Figures 4.12 to 4.15.
The signal–to–noise ratio and analytical range were calculated for each combination. The individual results for the detection limit are shown in Figure 4.17 and for the analytical range in Figure 4.17. The best result was received for the combination of SMI35 50001 with polyclonal rabbit anti–NfH
1
10000 (signal–to–noise = 9.1; analytical range = 73) which was therefore
CHAPTER 4. DEVELOPMENT OF A NEUROFILAMENT ELISA 95 1.7 1.2 1.2 1.0 1.0 2.0 8.6 2.1 1.4 Signal:Noise Ratio 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 ANTIBODY ANTIBODY NE14 SMI310SMI311 SMI32 SMI33 SMI34 SMI35 SMI37 SMI38
Figure 4.10: The detection limit of the NfH assay as determined by the signal–to–noise ratio (OD of the lowest standard divided by the blank OD) for different capture antibodies. A signal–to–noise ratio of greater than 1 (reference line) is required in order to obtain a readable signal.
CHAPTER 4. DEVELOPMENT OF A NEUROFILAMENT ELISA 96 Analytical Range 0.0 10.0 20.0 30.0 40.0 50.0 ANTIBODY ANTIBODY NE14 SMI310SMI311 SMI32 SMI33 SMI34 SMI35 SMI37 SMI38
Figure 4.11: The analytical range as determined by the signal–to–noise ratio for different capture antibodies for NfH.
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Figure 4.12: Optical density (OD) for different antibody concentrations of the polyclonal rabbit anti NfH antibody (second antibody) with the first antibody (SMI35) diluted 10001 .
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Figure 4.13: Optical density (OD) for different antibody concentrations of the polyclonal rabbit anti NfH antibody (second antibody) with the first antibody (SMI35) diluted 50001 .
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Figure 4.14: Optical density (OD) for different antibody concentrations of the polyclonal rabbit anti NfH antibody (second antibody) with the first antibody (SMI35) diluted 100001 .
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Figure 4.15: Optical density (OD) for different antibody concentrations of the polyclonal rabbit anti NfH antibody (second antibody) with the first antibody (SMI35) diluted 1000001 .
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Figure 4.16: Detection limit as determined by the signal–to–noise ratio for NfH 0.025 ng/mL. Columns are “colour”–coded (black, grey, white) accord- ing to the concentration of the second antibody as indicated in the legend. The absolute value of the signal–to–noise ratio is given for each antibody combination below the appropriate column.
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Figure 4.17: Analytical range as determined by the signal–to–noise ratio for NfH 1 ng/mL. Columns are grouped according to the dilution for SMI35 as capture (first). Columns are “colour”–coded (black, grey, white) accord- ing to the concentration of the second antibody as indicated in the legend. The absolute value of the signal–to–noise ratio is given for each antibody combination below the appropriate column.
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NfL assay The antibody concentration for the NfL assay was determined in a similar way to the NfH assay (data not shown). The optimal com- bination was found to be 1
5000 for NR4 and 5001 for the polyclonal rabbit
anti–NfL.