Chem 8157

6. Based on this information, the SAH-carboxyfluorescein-antibody complex should tumble more slowly than the unbound SAH-carboxyfluorescein. The equation for polarization should give values for P between 0 and 1. For instance, if a very large fluorescein-tagged molecule was rotating very slowly, we would only see a signal for fluorescence parallel to the excitation plane. Therefore, F_parallel would be 1 and F_perpendicular would be 0 (relatively), and our value for polarization would be 1. If we were observing the fluorescence from a molecule rotating very quickly (e.g. fluorescein by itself), then we would see an equal signal for fluorescence parallel to the excitation plane and perpendicular to the excitation plane: F_parallel and F_perpendicular would be equal, and we would get a value of 0 for P. Therefore, the scale on the y-axis doesn't make sense if the given equation was used to calculate polarization, but the trend still fits the expected results.

6.Based on this information, SAH-Carboxyfluorescein-antibody complex should tumble more slowly because it is bigger.The maximum and minimum value for P would be 1 and 0,respectively, corresponding to composition of toally rapidly rotating small molecules and totally slowly rotating big molecules. This doesn't fit the scale given in the y-ais of Figure 16.9.
11. The equation would be y=x.
12.Slops means that the diference of results get from the new method and established method depends on the amount of analyte and the intercepts is the difference that is independent on the amount of analyte.
13. Similar molecules present in plasma are expected interferences,especially, methionine when it presents in high concentration.
Concentration of methionine would interfere with detecting 0.2µM HYC:
0.2/0.1*4.5mmol/L=9mmol/L.

6.Since SAH-carboxyfluorescein has a much smaller molecular weight than that of SAH-carboxyfluorescein-antibody complex, the latter should tumbles more slowly. Equation 16.36 does make sense. Actually it's in agreement with the polarization what we learned before. The maximum absolute value for P is 1, which means P can be either 1 or -1, under the extreme condition that the big molecules only give parallel or perpendicular fluorescence. The minimum absolute value for P is 0, when the small molecule rotates really fast and gives no difference fluorescence in direction. I think the equation is actually cool because we can not only know the composition ratio of perpendicular or parallel light but also get information on which kind is in dominant by the + or - in front of the figure.
Based on the discussion above, the curve in 16.36 doesn't make any sense, because the Polarization value can only varies between 1 and -1.
11. If "a new method and an established method that perform equally in all respects", that means the results from the new method which are represented by x should be always the same a s results from the established method which are represented by y, so obviously y=x.
12. The difference in slope from the four equations mean the established methods and the new method have deviation when measuring the analyte. For example, a slope bigger than 1 represents either overestimation of the old method or underestimation of the new method.
I think the difference in intercept represent some systematic error, such as the difference in background.
13. The first part of this question is that those similar molecules like L-Cysteine or L-Methionine in the plasme. The second part of the question, I don't think we can calculate it. The reasons are: 1. We don't have a reference value telling us above which causes interference, while in my opinion, 4.5mmol/L giving response as 0.1umol/L HCY already causes huge interference considering the real HCY is only 0.2uM. 2. We don't have the relationship among [methionine]-polarization-[HCY]. We don't even know it's a curve or a line (figure 16.9 is not correct), so it's hard to calculate without a quantitative equation.

For #13, I agree with Meng about the interferences. Cysteine and methionine are potential interferences due to these common amino acids being present in large combination. If the enzyme was not specific for the reaction of free HCY -> SAH, there could be a reaction with the sulfur-containing amino acids and the efficiency of the assay could be dramatically decreased. It was determined that the antibody reaction is highly specific in the regards that the monoclonal antibody will not bind to the amino acids.
Meng, what is your reference about Fig. 16.9 saying it is incorect? Since this curve was produced with known standards of HCY, wouldnt deviation from this curve correlated with increased [methionine] or [cysteine] lead us to a conclusion of interference based upon the presence of the sulfur containing amino acid?

6. I agree with Meng that the polarization should be between -1 and 1. But I think that the Figure 16.9 do make sense because it don't show the unit of polarization. But the trend of the curve do make sense. Since the SAH-carboxyfluorescein-antibody complex is a bigger molecule, it tumble more slowly and has a bigger polarization. So when you add more HCY, the antibidy binds more to SAH instead of fluoresceinated SAH analogue. So in this case, less SAH-carboxyfluorescein-antibody complex exists in the system and less polarization is observed.
11. I agree with Li and Meng, when the two method give the same performance, x=y.
12. The different method is done by different labs. So the difference in slope may be caused by matrix effect. Since different labs use different HCY standard, maybe there are different interference concentrations or they use difference range of HCY concentration. Also, I agree with Meng, it may be an overestimation or underestimation situation. The intercept may somehow represent the background of the established method compared to the new method. If it is positive, the established method has a higher background; if it is negative, the established method has a lower background.
13. The interferences are some amino acids like methionine, because amino acids can react with adenosine to form a fluorescein or fluorescein-antibody complex. Generally, it behaves like SAH.
4.5mmol/L of methionine give the same signal as 0.1µM of HCY. If one want to control the interference effect less than 10%, the concentration of methionine should be 4.5* 0.2/0.1*10%=0.45mmol/L.

6.SAH-carboxyfluorescein-antibody complex is larger than SAH-carboxyfluorescein. So the former tumbles much slower and produces more emission. I agree with Greg Wolken's opinion. P ranges from 0 to 1. If it's a large molecule, P is 1 and if it's a small molecule, P is 0.From Figure 16.9, the y axis does not make sense.
11. If the two methods perform equally in all respects, the concentration of HCY we get from the same x using two methods should be the same. So y=x.
12.In my opinion, the difference in slope means the concentration of HCY we get using four methods are not the same. I think it can be caused by different agent(like HCY). The difference in intercept means there exist some fixed error between two methods. Maybe it is caused by different instrument, different agent except HCY and so on.
13.Since L-Cysteine and L-methionnie is similar with the structure of HCY, they can be interferences in the HCY assay. We only know 2 values of cocentration of inteference and HCY. I think it's not enough to predict the concentration of methionine interferes with detecting 0.2 uM HCY. We need more detailed information, if we had a plot of the relationship between concentration of HCY and methionine, it's much better.

For # 6
I find some information from Invitrogen website. P values ranges from –0.33 to 0.5. The reason is that F|| and F are interchangeable quantities and only differ in their normalization. For the Y axis in Figure 16.9, I think the problem is the unit. The figure should give unit. If the unit is mP (mp=P/1000), the values in the y-axis make sense.
Reference: http://probes.invitrogen.com/handbook/boxes/1572.html

SUMMARY AND COMMENTS

6. The axis of Figure 16.9 is not correctly labeled. It seems that these values are multiplied by 1000. We would need to go to the original reference, given at the bottom of Figure 16.9 to solve this mystery. The overall trend given in the figure is correct, though: (1) high difference in polarization for large fluorescent species (antibody + SAH-carboxyfluorescein), when [HCY] is low in the sample. (2) low difference in polarization for small fluorescent species (free SAH-carboxyfluorescein) when [HCY] is high in the sample.

The maximum and minumum values rarely match those that are predicted because the selection of polarized light is not 100% efficient.

11 and 12. The ideal equation is y=x when the signals in the blanks of the two methods being compared are different from zero. This is usually not the case. The intercept usually reflect this differnence. The slope can quickly tell us if a new method (Y) tends to overestimate (slope>1) or underestimates (slope

13. The interferences are methionine and cysteine. The former may likely form a complex with the antibody (compare the molecular structure of HCY and methione). The latter may interfere by reacting with adenosine in the presence of SAH (same chemical functionality).

Those that commented are right, that it is difficult to exactly calculate how much methionine will interfere with detection of 0.2 µM HCY. However, Jing is correct. One can use the information, given in the text, describing that 4.5 mmol/L of cysteine give a signal corresponding to 0.1 µM HCY.