Measuring the cotinine level in the serum of
smokers, non-smokers and passive smokers
using competitive ELISA
TOXICOLOGY
2013-12-13
(Version II)
Master program of biomedicine
Karolina Minta ()
Supervisor: Karin Lilja ()
ABSTRACT
Tabacco smoke is a general factor, which cause adverse effects on health affecting the heart, lungs and liver. The major ingredient in tobacco is nicotine, though much better biomarker of tobacco smoke is cotinine, stable metabolite of nicotine. The main method used in this research is competitive ELISA based on a competitive binding process between HRP-labeled cotinine and serum cotinine for the antibody binding sites. The main aim of the research was to measure and compare the level of cotinine in serum of smokers, passive smokers and non-smokers. Despite those three study samples, to the well strip coated with anti-cotinine antibody were also added: negative and three positive calibrators and serum sample as a negative control. According to the results from absorbance of the serum and calibrators it is possible to assess that in my sample C belongs to smoker, A to passive smoker and B to non-smoker.
INTRODUCTION
Cotinine is a primary, stable metabolite of nicotine produced in the liver. It is used as an indicator of tobacco smoke (including secondhand smoke exposure). (Florescu et. al.,2009)
The reason that the measurement of nicotine is replaced by cotinine is the difference in half-life. Nicotine have only 30 minutes, while cotinine can reach even 30 hours. This means that the level of cotinine remains stable in organism for a long time (even one week after tobacco use). In this research cotinine is used as a reliable biomarker to show the relationship between cigarette smoking and disease progression. (Kajsa, 2013)
Competitive ELISA relies on the competitive binding process between antigens (sample and add-in antigen). In this case the contest is between HRP-labeled cotinine and serum cotinine for the antibody binding sites (primary capture antibody bounded to the wells). The results of competitive ELISA are seen spectrophotometrically thanks to visualizing reagent, which detect the enzyme conjugate. Positive reaction does not give colour, while negative reaction is visible. The advantages of using competitive ELISA are high specifity, flexibility and it is suitable for complex samples. (Sino, 2011)
Competitive ELISA is used to measure the level of cotinine in serum of three different groups of people: smokers, passive smokers and non-smokers.
The main aim of this research was to show that antibodies are a good indicators for detecting given biomolecules, in this case cotinine. (Kajsa, 2013)
MATERIALS AND METHODS
Materials used In the research: Cozart EIA Cotinine Serum Kit
The experiment was performed on the 8-well strip coated with anti-cotinine antibody. The same amount of following samples were added to the well-strip: negative calibrator, three positive calibrators (with the concentrations amount to 10, 25 and 50 ng/ml of cotinine respectively), serum sample (as a negative control, because it has no cotinine) and three study samples (from smoker, passive smoker and non-smoker). Calibrator is used to describe the results as a positive or negative. The positive result must have an absorbance smaller or the same as calibrator with concentration of 25 ng/mL, while the negative should have bigger result of absorbance than calibrator 25 ng/mL. (Cozart, 2005)
Next enzyme conjugate, which was HRP-labeled cotinine, was added to each well. During incubation process HRP-labeled cotinine competed with serum cotinine for the anti-cotinine binding sites. The less amount of serum cotinine, the bigger possibility for HRP-labeled cotinine to bind to anti-cotinine antibody. The wells were washed with wash buffer to remove any excess enzyme material. Nonspecific or unbound components were washed away. If this step is missed, then antibodies that are aspecifically bound disturb the process of the research giving inconsistent results. Next 3,3’,3,3’-tetramethylbenzidine (TMB) was added to each well. It is a substrate solution, which allows to detect the horseradish peroxidase activity producing a visible signal. After incubation process the stop solution, namely sulphuric acid, was added for terminate the process. Stop solution is corrosive. If this solution is not added, then the substrate reaction will be not inhibited, what in consequence leads to saturation of the signal and the intensity of blue color will keep developing. The last step was to measure and read the absorbance spectrophotometrically at 450 nm.
RESULTS AND DISCUSSION
Figure 1.shows a standard curve of my results of the absorbance measured in different concentration of calibrator, respectively 0, 10, 25 and 50ng/ml.
According to the standard curve, with increase of calibrator concentration, the amount of absorbance falls. For 0 ng/ml the absorbance reaches 1,408, for 10ng/ml – 0,588, for 25ng/ml – 0,343 and for 50 ng/ml – 0,236. In accordance with the results from absorbance and based on absorbance of the serum it is possible to assess which study sample belongs to smoker, passive smoker and non-smoker.
Figure 2. shows the absorbance measured in different concentration of calibrator, from up 50, 25, 10 and 0ng/ml, the absorbance of my A-C samples and serum.
Serum achieved the absorbance equals to 0,429. Study samples A, B and C: 0,048; 0,399 and 0,047 respectively. The highest amount of absorbance received sample B. Bigger absorbance corresponds to less amount of serum cotinine that HRP-labelled cotinine can bind to antibodies and can be detected by TMB producing a colour. It is also clearly seen that B sample had the most similar amount in comparison to serum. Moreover, the negative control should have an absorbance bigger than 25ng/mL calibrator (0,343). Human serum is a negative control. Base on the absorbance of sample B (0,399) it is possible to read the cotinine concentration from the figure 1. It achieves approximately 19ng/mL. All those comparisons makes the sample B as a negative result, what means that it belongs to non – smoker person. The absorbance of sample A and B are lower than the absorbance of calibrator 50ng/mL, that is why the cotinine concentration in this samples cannot be read from the figure1. Sample C received the smallest absorbance, what means that many serum cotinine blocked antibodies precluding HRP-labeled cotinine to bind to them. Sample C belongs to smoker. Sample A has bigger absorbance than C and smaller than B, what corresponds to passive-smoker.
Figure 3. shows a standard curve of average results from the group 1 of the absorbance measured in different concentration of calibrator, respectively 0, 10, 25 and 50ng/ml.
The previous measurement of absorbances in different concentrations of calibrator are highly similar to the average results from the group 1 showed on the figure 3. This confirm that quality control is correct.
Figure 4. shows a standard curve of average results from the group 2 of the absorbance measured in different concentration of calibrator, respectively 0, 10, 25 and 50ng/ml.
In comparison to the average results from the group 2 the results were highly different (the absorbance of group 2 achieved smaller values). Such a difference could take place because different plate readers where used between those two groups.
According to the average results based from the blind samples and serum (with absorbance equals to 0,429), the sample of non-smoker is described as X. It is that because the value of X absorbance achieved 0,39 and by reading from the graph of average results from group 1 it is possible to see that the concentration of cotinine in the sample X is lower than 25 ng/mL calibrator and it achieves around 12 ng/mL. Y and Z samples achieved the absorbance of 0,05 and 0,048 respectively. Both of those results show in the graph (figure 3) that the concentration of cotinine in them is much bigger than 25ng/mL and it exceed the graph (the value is so big that it is impossible to read from the graph the exactly concentration from both samples). It means that Y and Z were exposed to tobacco smoke. The lower value of absorbance, so the higher concentration of cotinine belongs to sample Z, smoker. Thus passive smoker is Y.
CONCLUSION
To sum up, the cotinine is a reliable biomarker of tobacco smoke, which can be measure using test ELISA. It bases on the competitive binding process between antigens (HRP-labeled cotinine and serum cotinine) for the antibody binding sites. The results of test can improved our awareness of how big amount of this toxic substance can be present in smoker or passive-smoker.
REFERENCES:
Cozart (2005) Cozart EIA Cotinine Serum Kit M155B1/5. Available at: http://www.peramed.com/components/com_siparis/docs/M155B1_5060184000529_EN.pdf [collected 2014.01.03]
Florescu A., Ferrence R., Einarson T., Selby P., Soldin O., Koren G. (2009) Therapeutic drug monitoring. Methods for quantification of exposure to cigarette smoking and environmental tobacco smoke. (p. 364-370) Wolters Kulwer
Rabbaa-Khabbaz L., Daoud R.A., Karam-Sarkis D. (2006) A simple,sensitive and rapid method for determination of cotinine. Journal of Chromatographic Science.
Sino Biological Ins. (2011) Competitive ELISA: Basic Principles. Available at: http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/competitive-elisa [collected 2014.01.02]