Experimentation

VARIABLES

Independent Variable: The amount of histamine standards put into each vial.

Dependent Variable: The increase or decrease of the histamine levels with the addition of Turmeric and Mephyton(Vitamin K).

Control group: The vials of flies that have not been affected by either Turmeric or Mephyton(Vitamin K).

PURPOSE

The purpose of this experiment is to determine whether Mephyton(vitamin K) or Turmeric either help increase or decrease the levels of histamine in Drosophila fruit flies.

HYPOTHESIS AND RATIONALE

It is predicted that Turmeric powder and Mephyton(Vitamin K) will increase the levels of histamine because both of these substances carry anti-inflammatory inputs, which will then result in less inflammation all over the bodies of the Drosophila flies.

Mephyton(Vitamin K) and Turmeric are reported to be involved in reducing inflammation. Vitamin K has been involved in both cardiovascular and bone health, and recently in the movement of pro-inflammatory cytokines. Turmeric,on the other hand, can help get rid of Eicosanoids, which are molecules that promote inflammation in the body. Turmeric contains a compound called Curcumin that could help eradicate the Eicosanoids from the body.

INTRODUCTION

Rheumatoid Arthritis and related diseases have been around since organisms developed joints.

The immune system which usually defends itself, in turn attacks the joints in RA, making it an autoimmune disease.

In RA, harmed tissue mast cells of the immune system, for example the white blood cells and blood plasma proteins, leak from the bloodstream through the vessel wall and move to the site of damage, where they battle the contamination and recuperate the harmed tissue.

In disease such as RA, the immune system discharges an abnormal state of histamine, prompting the synovial coating around the joints to inflame.

To determine the levels of histamine in the Drosophila fruit flies, a Histamine ELISA Kit will be used. This protein connected immuno-sorbent examine, is a test that distinguishes and measures antibodies in the blood. This test can be utilized to decide whether one has antibodies identified with certain irresistible conditions. Antibodies are proteins that the body creates in light of unsafe substances called antigens(Kinman, 2017).

DATA ANALYSIS

In order to get the standard curve from the absorbance reading, what I needed to find was the maximal binding value,which would be the averages of the standard concentration which was run in duplicates. Since the first standard concentration was 0ng/mL, I took the absorbance values from that set, added them, and divided them by 2. The number is then to be divided by itself, knowing that that would be the initial standard value for 0ng/mL. Then, in order to get the percent value, the number was multiplied by 100. This was calculated as follows:

Percent of Maximal Binding for 0ng/mL:

Values from the set: 1.878 + 1.547 = 3.425

3.425 / 2 = 1.7

1.7 / 1.7 = 1 * 100 = 100%

This process was then repeated for all the other standard concentrations.

After getting the Percent of maximal binding for the standards, what I had to figure out next was the maximal binding for the average values of the Control, Turmeric, and Vitamin K that I got from the plate reader. So, the way that I calculated the %B/BO was to first divide 0.3526, which was the average value for the Control and divide it by 1.7. After that, in order for it to yield the percentage, I simply multiplied the number by 100; to get 21%. The calculations goes as follows:

Average Value from plate reader for Control: 0.3526

0.3256 / 1.7 = 0.2074

0.2074 * 100 = 21% == %B/BO

This process of calculation was then repeated again with the average values of the Turmeric and Vitamin K.

Last, I needed to figure out, for what standard concentration was the maximal binding at. For this particular calculation, I needed to use the equation of the line of best fit. The %B/BO for the Control, Turmeric, and Vitamin K were all y-values in the equation, which meant solving for the x-value to get the standard concentration. The calculation goes as follows:

Equation of the Line = y = -1.25x + 65.1

Plug in 21 (Control) for the y-value and solve for x:

21 = -1.25x + 65.1

- 44.1 = -1.25x

X = 35.28 ng/mL for the maximal binding at 21%

This process of calculation was then repeated for Turmeric and Vitamin K.

According to the data that was received, there is a trend that shows both the Vitamin K and the Turmeric actually increased the histamine levels in the fruit fly, which was indicated as a really light blue color as compared to the standard, therefore resulting the presence of histamine; whereas if histamine wasn’t present, it would result in a pigmented blue color.

METHOD

Part 1: Preparing the food with Turmeric

In a weigh-boat, measure out 5.13 grams Fruit Fly food.
In a separate weigh-boat measure out 0.45 grams of Turmeric and mix it with the Fruit Fly food. Transfer the food into vials and add water until the mixture absorbs it and makes a paste.Mark these vials as Turmeric.
To transfer the flies into the vials, put them in the freezer for about 3-5 mins. This process the flies to go into a sleep mode. Carefully transfer the flies into the vials with the turmeric.

Part 2: Preparing the food with Mephyton(Vitamin K)

In a weigh-boat, measure out 5.13 grams of Fruit Fly Food.
Take a beaker and fill it up with 10 ml of water.
Take a syringe and withdraw 1 ml of Mephyton(Vitamin K), and insert it into the beaker. Mix well until the Vitamin K dissolves into the water.
Mix this solution with water until it turns into a paste-like consistency.
To transfer the flies into the vials, put them in the freezer for about 3-5 mins. This process the flies to go into a sleep mode. Carefully transfer the flies into the vials with the turmeric.

Part 3: Reagent,Wash Buffer, and Dilution Buffer Preparation

Bring all reagents, plate wells to be used samples and calibrators to room temperature(20-25 degrees Celsius) before use.
Prepare the necessary volume of wash buffer by mixing 1 part 25X Wash Buffer with 24 Deionized water. Label as Working Wash Buffer.
Mix entire contents of foil pouch in1 liter deionized water. Label as Dilution Buffer.

Part 4: Assay Protocol

Add 50 ul of standards or diluted (if necessary) sample to the appropriate wells.
Mix each reagent by inverting the reagent bottle prior to use.
Add 50 Mu/L of the Histamine-HRP Conjugate to each well. Use a multi-channel or repeater pipette if appropriate.
Mix by gently shaking the plate. A microplate shaker may be used if available.
Cover the plate with plastic film and incubate at room temperature (18-30 degrees Celsius) for 45 minutes.
Empty out the contents of wells into sink and blot on paper towel to remove as much fluid as possible.
Add 300Mu/L Working Wash Buffer per well.
Shake plate slightly during soak period for best wash results.
Empty wash solution into sink by inversion then blot plate against clean paper towel to remove any remaining washing buffer.
Repeat steps 7-9 for 3 times.
Add 150 Mu/L of substrate to each well. Be careful not to touch the inner well walls. Use a multichannel pipette for best results. Mix by shaking plate gently.
Incubate at room temperature (18-30 degrees Celsius) for 30 minutes.
To ensure uniform color development, before recording absorbance gently shake the plate by sliding it back and forth on a flat surface or, if available, use the shaker function on the reader.
Measure the absorbance at 650 nm if not using Stop Solution else, add 75 Mu/L Stop Solution (1N HCL).... to each well then measure the absorbance at 450 nm.

EXPERIMENTAL ERROR

There is a possibility of the occurrence of human error in the form of an inaccuracy while transferring the Substrate and the Histamine Enzyme Conjugate into the wells. The scientific procedure and safety precautions were followed throughout the experiment. The materials that were used throughout this experiment have been thoroughly cleaned, sterilized and gloves were worn on at all times. Special care was taken during the incubations and while transferring the certain liquids to the wells. Also, the precaution was taken of not allowing any liquids from the ELISA kit to be transferred into glassware. Also, the experiment that was done was in duplicates, in order to rule out the possibility of inaccurate data, in the form of results.Thus, an extra effort was made to avoid experimental error.

CONCLUSION AND IMPACT

In conclusion, the levels of histamine did increase in both samples of Vitamin K and turmeric. My hypothesis stated, “Turmeric powder and Mephyton (Vitamin K) will increase the levels of histamine because both of these substances carry anti-inflammatory inputs, which will then result in less inflammation all over the bodies of the Drosophila flies.” My hypothesis was proven right because the maximal binding of the Turmeric and Vitamin K displayed the levels of histamine increase as compared to the Standard. But that was not the main objective of this experiment. Although it was previously stated that turmeric and vitamin K carried these anti-inflammatory inputs, these substances do not prove to reduce or lower the inflammation in the flies. On the other hand, RA is an autoimmune disease, which practically means that the system is self-destructive. In this disease, instead of histamine being mass produced, we want to contain the histamine levels in a way such that it does not inflame the sinovial coating around the joint. Like I indicated earlier, figuring out a certain medication to stop the release of histamine and prevent inflammation, we would be able to help millions of people that have been diagnosed with RA cure their disease and maybe provide a safe chance for recovery.