A substrate is decomposed in the presence of an enzyme according to the Michaelis-Menten equation
with the following kinetic parameters:
Km = 20 g/L
Vmax = 12.0 g/L-min
(a) Determine the concentration of substrate after leaving the second reactor in a two-reactor series of
20-liter CSTRs. The flow rate is 2.00 L/min. The inlet substrate concentration is 40 g/L. The
enzyme concentration in the two reactors is maintained at the same value all of the time.
(b) Determine the conversion in terms of substrate conversion of the two-reactor system and the
conversion of a single CSTR equal to the total volume of the two-reactor system. Clearly indicate
the configuration with the greater conversion

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA)

At the conclusion of this exercise you should be able to:

• explain the principles of the enzyme-linked immunosorbent assay (ELISA).
• determine the qualitative results of a clinical scenario using ELISA.
• understand the purpose of the reagents used in ELISA.

Background

Immunology is the study of the immune response. This involves the interaction between antigens, antibodies, and cells. Immune responses are generated by the body to recognize substances considered to be “foreign”. Microorganisms can generate different immune responses when they infect a host. This leads to the formation of antibody in a host. This exercise will explore some of the basic principles of immunology using an indirect ELISA to identify the presence of antigen in a sample of serum.

Scenario

A serum sample was obtained from a patient who was affected by a severe gastrointestinal infection, which was presumed to be caused by a bacterial infection. In order to investigate the causative agent, an ELISA using antibodies of known intestinal pathogens was performed to identify the microorganism that caused this infection.

Materials

1. A sample of serum was obtained from the child.
2. Antigens obtained from three known gastrointestinal pathogens:

Shigella sonnei, Escherichia coli, and Salmonella typhimurium

3. Microtitre polystyrene plate.
4. Coating buffer (phosphate buffer solution, pH 9.6; PBS).
5. Blocking buffer (PBS/Tween)-Tween is a detergent added to buffer to prevent non-specific binding of molecules.
6. Secondary antibody (anti-human immunoglobulin). This antibody is attached to an enzyme that will react with a substrate that is added in the last step of the process.
7. Enzyme substrate: this is used in immunoassays. For this assay, the substrate used is horseradish peroxidase, which causes a yellow reaction once it attaches to the secondary antibody.

Procedure

1. Add 50 µl of each of the three antigens diluted in coating buffer to rows A-C and columns 1-12 of a microtiter plate.

Row A: Shigella sonnei

Row B: Escherichia coli

Row C: Salmonella typhimurium

2. After incubating for 1 hour at room temperature remove liquid by tapping onto paper towel.
3. Add 300 µl blocking solution to each well and leave for 5minutes.
4. Tap out residual solution.
5. Add 50 µl of PBS/Tween to rows A-C and columns 1-12.
6. Add 50 µl of the patient’s serum to column 2 and rows 1-3.
7. Proceed to make two-fold dilutions in each row by removing 50 µl from column 2 and transferring to column 3. Continue until the last column is reached (column 12).
8. Incubate the plate at room temperature for 1 hour.
9. Empty plate by tapping out fluid on a paper towel.
10. Fill each well with wash solution (PBS/Tween), invert and remove residual liquid. Repeat 3 times.
11. Add 50 µl of secondary antibody to each well and incubate 1 hour at room temperature.
12. Remove residual liquid as described above and complete 3 washes as above.
13. Add 50 µl of substrate to each well and note the change in colour in each well.

Results

The results of this experiment are shown below.

Questions

1. Which organisms was the causative agent of infection for this patient? Explain you answer in comparison to the other samples provided.

2. What is the purpose of using a negative control? Explain what it means of column 1 wells appear yellow.

3. What are the fundamental differences between direct and indirect ELISA methods?

4. Explain the purpose of diluting the serum sample provided.

5. Describe the purpose of each of the components in the ELISA.

Assume that you identify a set of conditions under which glycolysis and gluconeogenesis occur in parallel and at the same rate. What do you expect in terms of the net energetics of this system?

Group of answer choices

neither gain nor loss of high energy bonds

loss of four high energy bonds

gain of three high energy bonds

gain of one high energy bond

loss of two high energy bonds

Consider the following inter-conversion, which occurs in glycolysis: Fructose-6-phosphate ↔ Glucose-6-phosphate Keq’ = 1.97 What is ΔG°’ for the reaction (Keq’ measured at 25°C; and R=8.314 J•mol-1•K-1)? (b) If the concentration of fructose-6-phosphate is adjusted to 1.5 M and that of glucose- 6-phosphate is adjusted to 0.50 M, what is ΔG? (c) Why are ΔG°’ and ΔG different?

1. In the boxes in the flow chart below, follow the flow of carbon atoms in the complete oxidation of glucose by indicating the beginning and ending molecules in each of the steps of cellular respiration starting with 1 molecule of glucose. Be sure to include the number of molecules of each type of molecule made from 1 molecule of glucose at each step. (Do not include other molecules made like NADH and ATP).

                               Glycolysis                 Pyruvate Processing               Citric Acid Cycle

Many molecules play multiple regulatory roles in cellular processes. Give an example of a molecule that plays more than one role in regulating the reactions we've learned about this week, and explain why it acts in the same OR a different manner in each instance.

The molecule I am choosing is pyruvate but what other reactions does pyruvate regulate besides glycolysis and how? please help, I am very confused.

Human Resource Planning (HRP) represents a significant improvement on traditional Manpower Planning exercise.

To what extent does HRP represent an improvement towards an organisation’s assessment of it human resource needs?

1.0 Introduction

2.0 Human Resource Planning (HRP)

     2.1 Definition of Human Resource Planning

     2.2 Role of Human Resource Planning

     2.3 Objectives of Human Resource Planning

      2.4 Process of Human Resource Planning

The building blocks of fats are called fatty acids and contain a lot of carbon atoms. They vary depending on a little structure called a double bond.
Let’s start by talking about the role of fat in the diet

Which of the following pairs of acids are incorrectly ordered?

stronger acid                weaker acid

a) HF                               HI

b) PH3                            NH3

c) HF                               NH3

d) H2S                            H2O

e) H2O                           NH3