Glucose and Insulin ELISA
Name
Institution
Course
Tutor
Date
Abstract
Glucose homeostasis is the process by which human bodies maintain ideal concentrations of blood glucose. The process is important in providing continuous supply of energy for proper body functions. Abnormal glucose homeostasis in diabetic subjects may result to long-term health problems and this may lead to severe damage of vital organs in the body. In the experiment, The Oral Glucose Tolerance Test (OGTT) is used to illustrate the changes in blood glucose after consumption of glucose after 12 hours fast.
Introduction
The paper analyses glucose homeostasis for a period of 2 hours after consumption of glucose. This is after 12 hours of fasting. The hormones responsible for the blood glucose and regulations are glucagon and insulin. In this case, the effects of glucose concentration increments on Insulin Enzyme-Linked Immunosorbent Assay (ELISA) were examined (Accorsi, 2009). The experiment helps to obtain the relation between dilution and optical density of glucose. In so doing, the experiment helps to calibrate Mercodia Mouse Insulin ELISA (a two-site enzyme immunoassay). It also helps in calibrating Mercodia Mouse Insulin ELISA and hence obtaining the relevant equation (Komatsu et al. 2006).
Data collection Methods (glucose level)
Before the experiment, fasting was observed for 12 hours (overnight). The fasting was to ensure that majority of the glucose in the body were used up. Though the glucose could not be reduced to zero (after fasting for 12 hours) but fasting ensured it was reduced to the lowest quantities. The high glucose levels in the body could not give a better reflection of the effects of consuming glucose on the blood sugar, mars bar and cereal bar.
In the practical 10 students’ blood were tested. Four students were tested for glucose solution. Three students were tested for Mars bar and other three students tested for cereal bar. The hands were washed and dried before the experiment (taking the blood samples). The goal of testing laboratory was to maintain the highest level of accuracy and precision in the end results and so the reason for drying the hands. Picking the students tested for a particular concentration was done randomly to minimize result biasness.
During the experiment, a control blood sample (about 100 μl – two large drops) was withdrawn by the help of the Glucoject Dual overleaf. The mean glucose level was obtained. Normally, measurement errors occur while collecting the measurements. The control group was essential in comparing the results. This might result from use of wrong equipment in measuring samples, poor calibration of the equipment and taking wrong readings. The equipment can be so used up such that the calibrations are not clear. The person taking the readings might thus end up recording the wrong reading resulting to an error in the final results. The use of Glucoject Dual overleaf ensured accurate results.
After taking the first sample, 75 g glucose was consumed in the form of glucose solution. The solution was given to all the ten equally and at the same time. They ate nothing thereafter.
One hour later (after glucose consumption), blood samples were taken after 1 hour and the next sample after two hours (from consumption time). In each sample, the blood glucose was measured and plasma collected. This was to target the peak of blood glucose which usually occurs between 30 minutes and 1 hour in normal cases. However, in diabetes cases, it occurs closer to 2 hours. No diabetic student’s blood was sampled.
The results are shown in the Appendix, table 1.0.
Data collection Methods (Insulin Enzyme-Linked Immunosorbent Assay (ELISA))
10 μl of each of your samples pipetted into well (2 wells used).
Into each of your 2 wells 100 μl of enzyme conjugate 1x solution was added.
On a plate shaker (700-900 rpm) enzyme was incubated for 2 hours at room temperature. The room temperature ensured good temperature for the action of enzymes.
The reaction volume was discarded by inverting wells over a sink. 350 μl wash buffer 1x solution was added each well. The wash solution was discarded by firmly tapping absorbent paper to remove excess liquid severally. This was repeated five times.
200 μl Substrate TMB was added to each well.
It was then incubated for 15 minutes at room temperature. The time was sufficient for the enzymes to react.
50μl Stop Solution was added to each well. The plate was then placed on the shaker for approximately 5 seconds to ensure mixing.
The optical density at 450nm was read. This was before 30 minutes elapsed.
The calibrated results were used to draw a standard curve useful in working the insulin concentration on samples. The curve helped in determining the relation between dilution and calibrators.
The graph on density against concentration was plotted to determine the equation connecting the two. The detection limit was ≤ 0.10 μg/l. The low level ensured high accuracy of the recorded data.
The results are shown in appendix table 1.2.
Discussions
The concentration of glucose is among the major factors determining the direction of its metabolism. Glucose concentration in the body is determined by various factors among its intake. The higher sugar intake leads to increased glucose level in the body and vice versa. Glucose concentration in the body is as well determined by the amount other related food intake and sugar concentration in the body is regulated by insulin. Consuming glucose leads to increased glucose level in the body as observed in the experiment.
A drop in the glucose content is accompanied by gluconeogenesis activation, as a rise in glucose concentration boosts glycolysis (the process through which glucose is broken down). In other words, the glucose level in the body is maintained by a type of negative feedback. In general, metabolic activities are regulated by changing the activities of some enzymes of the pathway. The one hour time was significant as it ensured that the glucose had been digested and absorbed into the blood stream. Glucose digestion takes a very short time since it I in its simplest form and does not require further breakdown.
Glycolysis is under hormonal (insulin) and allosteric control. Insulin activates glycolysis by stimulating key glycolytic enzymes and this is responsible for reducing the amount of glucose concentration in the body. The other method of reducing glucose in the body is fasting. During fasting the glucose in the body is consumed and when there is no consumption, the level reduces. Food taken (carbohydrates) enter the human body in complex forms like disaccharides, glycogen and polymer starch but this is not the case with glucose. The first metabolic step is the breaking down of bigger polymers to simpler, soluble forms that can be transported through wall so the intestines and delivered to the tissues. Glucose however, is in its simplest form (monosaccharide) and enters the glycolysis stages directly. This is the reason as to why the level when glucose is consumed, the blood sugar level increases shortly. The consumption of glucose in the solution form ensured and increased the level of glucose into the body.
The results of glucose concentrations show that the glucose levels, after consumption of glucose reduced after fasting but increased one hour after its consumption. The consumption increased the amount of glucose in the body resulting to increased glucose concentration but after glycolysis, the level is expected to reduce. The mean before glucose consumption was 5.45, then moved to 7.95 one hour after consumption and then reduced to 7.13 two hours later after consumption. Glycolysis that takes place soon after glucose consumption is the splitting of complex sugars to simple and soluble sugars. In the process glucose is broken down into two molecules each containing three-carbon sugar. Glycolysis produces two ATP molecules, two pyruvic acid molecules and two molecules of NADH. The process can take place with or without the presence of oxygen.
Glycolysis occurs in steps. The first step entails enzyme hexokinase phosphorylates which adds phosphate group to glucose in cytoplasm. The process leads to the production of glucose 6-phosphate. The chemical process is shown below.
Glucose (C6H12O6) + hexokinase + ATP → ADP + Glucose 6-phosphate (C6H11O6P1) and then glucose 6-phosphate is converted into isomer fructose 6-phosphate by phosphoglucoisomerase (enzyme). Isomers have similar molecular formula with the difference in arrangements of atoms. The chemical process is shown below.
Glucose 6-phosphate (C6H11O6P1) + Phosphoglucoisomerase → Fructose 6-phosphate (C6H11O6P1)
Another ATP molecule is uses by the enzyme to transfer phosphate group to fructose 6-phosphate to form fructose 1, 6-bisphosphate.
Fructose 6-phosphate (C6H11O6P1) + phosphofructokinase + ATP → ADP + Fructose 1, 6-bisphosphate (C6H10O6P2),
Fructose and 6-bisphosphate are further split to form isomers of each other. In short, one glucose molecule in Glycolysis gives about two molecules of pyruvic acid, two ATP molecules, two NADH molecules and two water molecules. The end equation is Glucose (C6H12O6) + 2 [NAD]+ + 2[ADP (Adenosine Diphosphate)] + 2 [P]i —> 2 [C3H3O3]-(Pyruvate) + 2 [NADH] (Reduced Nicotinamide Adenine Dinucleotide) + 2H+ + 2 [ATP] (Adenosine Triphosphate) + 2 H2O (Odensten et al. 2007). The energy and water lost gradually reduced the glucose level and hence low concentration levels finally. During the first one hour, insulin level remained low due to decreased blood sugar level. The glucose level in the blood was low and hence insulin level remained low. One hour after consumption, insulin level increased to decrease the raised blood sugar level and then decreased later. The glucose level rose in the body after one hour and so the need for increased glycolysis. This resulted to increased insulin production to speed up glycolysis.
Glucose, Mars bar and Cereal bar
The results of students Glucose, Mars bar and Cereal bar before and after fasting are recorded in table 1.0 (Appendix). The amount of glucose, mars bar and cereal bar different from one student to the other and so it was important to get the average. This was affected by various health conditions and the types of food eaten 12 hours before the fasting was observed. The average assumed a general characteristic of the blood samples taken. The mean of glucose was 0.15 at the start of the practical and 1.19 after one hour (after consumption of glucose). Mars bar had an average of 0.18 before and 0.59 after consumption of glucose. On the other hand, the students had a mean of 0.11 for cereal bars before and recorded 0.62 after glucose consumption. After consumption, in all the three variables, glucose, mars bar and cereal bar, their levels increased due to increase in the blood sugar. The mean of mars bar was the highest followed by glucose and then Cereal bar (before the consumption of glucose). After consumption, mean of glucose recorded the highest then cereal bar and Mars bar recorded the least. Consumption of glucose raised blood sugar and this increased the glucose in the body. The glucose increased to the highest followed by cereal and then mars bar after one hour. Glucose, being a monosaccharide was directly absorbed into the blood quickly increasing the glucose level to 1.19 as recorded. Mars bar and Cereal bar be obtained after the digestion of glucose and so their quantities did not increase as much as glucose. This is also observed before the consumption that the level of mars bar was the highest. They are obtained after process that took place slowly after the consumption and so the slow increase in their masses. All the sample students had glucose but some lacked mars bar and others did not have Cereal bar. This means that the amounts of glucose in the blood exceed the amount of Cereal and mars bar in the blood. The practical does not show any direct relations among the three.
The graph shows the increase in the three variables after and before glucose consumption.
Before glucose consumption After Glucose consumption
Glucose solution 0.15 1.19
Mars bar 0.18 0.59
Cereal bar 0.11 1.25
Elisa
Mercodia Mouse Insulin ELISA is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. At the time of incubation, insulin in the sample reacts with peroxidise-conjugated anti-insulin antibodies and anti-insulin antibodies bound to microplate wells. A simple washing step removes unbound enzyme-labelled antibody. The bound conjugate is detected by reaction with 3, 3’, 5, 5’-tetramethylbenzidine. The reaction was stopped by adding acid to give a colorimetric endpoint that is read spectrophotometrically.
Consumption of glucose leads to increased glycolysis. Insulin helps in the reduction of glucose levels in the body through glycolysis. At the incubation time, insulin samples reacted with eroxidise-conjugated anti-insulin antibodies and anti-insulin antibodies bound to microplate wells. The washing machine helped remove unbound enzyme-labelled antibody. The reaction of conjugate with 3,3’,5,5’-tetramethylbenzidine was stopped by adding acid.
The graph of ELISA is a straight line indicating dilution and optical density are directly proportional with a given constant. Due to measurement errors and inaccuracies, a straight line would not be obtained but this can be obtained considering the line of best fit. The line of best fit cuts the x-axis on the positive side 9and hence the constant 0.0197.
Graph of Calibrators against dilution
Letting optical density to be y and Dilution to be y, we have y = 0.3687x +0.0197 where y= mean of conditions and x = the unknown as evident in table 1.2 appendix. The constant is 0.0197. The detection limit was ≤ 0.10 μg/l and this enhanced accuracy. The practical hence is successful in determining the equation relating optical density and dilution.
Assumptions in generating the equation
It was assumed that all the students observed a fasting of 12 hours before the experiment. If the fasting was not observed, the test results are fault as they are unscientific. The entire participants were further assumed to have good health and none diabetic or any other related diseases. The equipment used was assumed to record data of the highest accuracy. Error in sampling can result from the equipment used, biased sampling as well as the materials used.
Conclusion
In conclusion, it was recorded that the levels of all the three variables increased one hour after glucose consumption. The practical assumes that the students never took any food 12 hours before the experiment and were of good health. None was diabetic or had conditions related to diabetes. Consuming glucose increases the blood sugar and so increase in the three; Glucose, Mars bar and Cereal bar. The variables that are obtained after glucose digestion increased slow as glucose had to undergo a process before they were formed. One hour after consumption gave enough time for glucose to be absorbed into the body and formation of Mars bar and Cereal bar. All the concentrations were measured form the blood samples. The glucose consumption lead to increased glycolysis as there resulted a need to control the blood sugar. Glycolysis had to increase to deal with the increased blood sugar. Major decisions are made based on these results and any form of error not only affects the laboratory, but also other sectors that apply that data. The line of best fit could not accurately determine the relation between dilution and Calibrators.
References
Accorsi, P., Govoni N., Gaiani R, Pezzi, C., Seren E., (2005). Insulin and Metabolic Parameters Throughout the Dry Period and Lactation in Dairy Cows. Reprod Dom Anim 40:217-223.
Komatsu, T., Kushibiki, S., Hodate, K., (2006). Effects of ghrelin injection on plasma
Concentrations of glucose. Comp Biochem Physiol 143:97-102
Odensten, M., Berglund, B., Waller, P., (2007). Metabolism and udder health at
dry-off in cows of different breeds and production levels. J Dairy Sci 90:1417-1428
Appendix
Results
Table 1.0: Glucose results
Conditions Fasting Mean Fasting After 1 hour Mean after 1 hour After 2 hours Mean after 2 hours
Glucose Solution 1 5.6 5.45 10.0 7.95 8.4 7.13
Glucose Solution 2 5.9 9.2 7.1 Glucose Solution 3 5.1 6.3 7.2 Glucose Solution 4 5.2 6.3 5.8 Mars bars 1 6.1 6.2 6.9 6.73 4.9 5.8
Mars bars 2 6.6 6.6 6.1 Mars bars 3 5.9 6.7 6.4 Cereal bars 1 6.7 6.27 5.3 7.2 5.4 6.07
Cereal bars 2 6.1 8.0 6.5 Cereal bars 3 6.0 8.3 6.3 Table 1.2: Results for Insulin ELISA
Dilution Calibrators
.2 µg/L 0.174
.5 µg/L 0.195
1.5 µg/L 0.437
3.0 µg/L 1.190
6.5 µg/L 2.416
Time Glucose Solution Mean The unknown
0 0.15 0.13 0.15 0.16 0.15 0.14 0.15 0.15 0.34
1 0.32 0.28 0.45 0.35 2.32 2.39 1.62 1.78 1.19 3.16
Mars bar Mean 0 0.12 0.17 0.29 0.16 0.17 0.18 0.44
1 0.16 0.61 0.49 0.88 0.81 0.59 1.54
Cereal bar Mean 0 0.11 0.12 0.11 0.11 0.11 0.11 0.11 0.25
1 0.14 0.15 0.42 0.49 0.16 0.14 0.25 0.62
1536704705350Table 1.3 Glucose solution
6864351181100