Enzyme assay pdf

If our mucosal surfaces were spread out they would cover an area equal to one and one-half tennis courts, so the importance of IgA in protecting our mucosal surfaces cannot be overstated. IgA can exist in four different forms: monomer, dimer, trimer or tetramer. However, the IgA form most common in external secretions, such as saliva, is called secretory IgA, and it is the dimeric form in which two IgA molecules are joined together by the J chain.

IgA is made by plasma cells in the mucosal lymphoid tissue and secreted through the epithelium into the lumen of the respiratory tract or gastrointestinal tract. The transportation of IgA across the epithelium into the lumen is mediated by a receptor that is synthesized by mucosal epithelial cells Fig. After synthesis and secretion by the plasma cells, dimeric IgA binds to the receptor on the mucosal epithelial cells to form a complex.

This complex is endocytosed into the epithelial cell and actively transported in vesicles to the luminal surface.

Here the receptor is enzymatically cleaved and the IgA molecule, with a portion of the receptor, is released into the intestinal lumen. Once in the lumen, IgA affords protection by mechanisms that include I interfering with microbial adherence to mucosal surfaces, II inhibiting penetration of antigens across the epithelial surface.

Studies have established the role of salivary IgA in limiting respiratory infections. Certain studies show a decrease in the saliva concentration of IgA in 2 athletes that has been proposed as a possible causal factor in the established 3 increased susceptibility of athletes to upper respiratory tract infections URTI.

The effects of exercise on s-IgA are therefore still under intense study. IgA deficiency is quite common, with approximately one in every people some studies show 1 in people presenting a deficiency of IgA.

Most of these people appear healthy and may never know that they have selective IgA deficiency. Conversely, there are people with IgA deficiency that have severe illnesses. It is yet unknown why some people with IgA deficiency are very sick while others are not.

It is therefore very important that during the ELISA assay student saliva be mixed between pairs of students in case there is an IgA deficiency in the student population. Sampling and collection of saliva is easy and non-invasive. This makes use of saliva acceptable even in non-laboratory settings. Consequently, the technique proposed is applicable in a high school setting.

References: 1. Gleeson M. T, McDonald W. Salivary IgA subclasses and infection risk in elite swimmers. Immunol and cell Biol. Laing S. Salivary IgA response to prolonged exercise in a hot environment in trained cyclists. Nieman D. C, Nehlesen-Cannarella S. The effects of acute and chronic exercise of immunoglobulins. Sports Med IgA images on page 1 were obtained from: The science creative quarterly issue 3, www.

ELISA is used in medicine and in quality control check in many industries. In the simplest form of ELISA, an unknown amount of antigen is bound to a surface, and an antibody specific to the antigen is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and a substance that can be converted into a detectable signal by the enzyme is added in the final step.

The quantity of antigen in the sample is determined by the signal intensity. This technique involves the following general steps: 1. Surface on which a known amount of capture antibody is bound. Non-specific binding sites on the surface are blocked. Antigen-containing sample is added onto the plate. Primary antibody that binds specifically to the antigen is added. Enzyme-linked secondary antibody that is specific to the primary antibody is added. The plate is washed so that the unbound antibody-enzyme conjugates are removed.

A chemical is added that is converted by the enzyme into a color or fluorescent or electrochemical signal. The signal is measured to determine the quantity of antigen in the sample. Goldsby, T.

Kindt, B. Osborne, Kuby Immunology, 4th ed. Freeman and Company, , p. Here, an unlabelled antibody is incubated in the presence of its antigen. The plate is washed so that unbound antibody is removed the more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence competition. The secondary antibody, specific to the primary antibody is added.

The secondary antibody is coupled to an enzyme. A substrate is added and remaining enzyme elicits a chromogenic or fluorescent signal. For competitive ELISA- the higher the original antigen concentration, the weaker the eventual signal.

Hence, the antigen concentration in this case the antigen is IgA is inversely proportional to the fluorescent signal. Adapted from www. Time Requirements Approximately 3 double period classes will be needed to complete all the lessons.

Day 1. In the second half of the class the students will play the secretory IgA game. Day 2- Assemble the simple spectrometer and perform the simple dilutions lab.

Day 3- Students will perform the sIgA lab. On the back of 25 of these questions write the word bacteria and on the other 25 write the word virus. These cards will be taped onto the shirts of respective group members during the activity. This will be taped onto the wall.

Dilution lab: Unknowns should be made ahead of time with student input. Label each sample clearly. These will serve as the unknowns. This is the unknown source that I used with my students.

The teacher should find an immunology laboratory for steps of this lab. A local university or college with an immunology department should have the necessary equipment.

This arrangement will need to be made well in advance of starting the lab, preferably at the beginning of the academic year. Students should be able to use a pipette before the lab. Determine the plate layout. The kit I recommend here can hold up to 76 samples. It is best to use one kit per class as the standards are limiting but with good organization, one kit can be used for two or more classes to cut on costs.

Go over the procedure and complete the labels for the tubes- this can be done with student participation. Explain to the students that each group of two students will calculate their average flow rate. The procedure is long, so discuss it with the students prior to the lab.

Select students that have mastered lab1 on simple dilutions to serially dilute the standards as a demonstration to the class. Prior to the lab- go over the procedure with these students. Prepare a chart- one for each class where all the data from the standards and samples will be written. You may use the plate layout as a guide.

This table will be taped on the wall and students will write down the values for their IgA concentrations on this table after interpolation from the standard curve. Materials and Equipments Secretory IgA class activity index cards with color variety.

Each group of two students will assemble one spectrophotometer. Therefore, 15 spectrophotometers needed for a class of 30 students.

You may adjust the group sizes based on parts availability. How to set up a simple spectrophotometer. Detailed information at www. You make take students with you to the lab after making the necessary prior arrangements. An introduction to radioimmunoassay and related techniques 4th Ed. Amsterdam: Elsevier. Students are also expected to have a general understanding of how the immune system works including the difference between the innate and adaptive immunity.

They should have good graphing skills and be able to compute simple algebraic equations. They should also be able to use pipettes to make accurate measurements. Students should know what optical density is and how it is used to determine concentrations of standards and unknowns during ELISA. Student Expectations 1. Play the IgA game and participate in the subsequent class discussion 2.

Complete the spectrophotometer assembly. Complete the simple dilution lab, construct a standard curve and use this standard curve to determine the concentrations of unknowns. Complete the SIgA lab and hand in lab sheet. The teacher will introduce these terms and explain their relevance to the subsequent lessons. Make cards to represent saliva, two molecules of SIgA showing a binding site you will have four students do this as this is a dimeric protein , 1 bacteria with the same binding site that corresponds to the SIgA , 1 virus, 1 IgA secretory receptor and one enzyme.

On the backs of these cards write down what each does. The cards should be color coded to make it easier for students to recognize them. Additionally, the teacher will have prepared 50 questions on immune system and written them on index cards, 25 questions labeled virus and 25 labeled bacteria. Students can be involved in the preparation of these questions. Sample questions: a Give two examples of organs that are involved in the immune response.

The process of sIgA synthesis and secretion. After synthesis by plasma cells, dimeric IgA passes through the mucosal epithelial membrane in a poly-Ig receptor mediated manner. One group will role play the pathway and answer questions to protect the respiratory membrane while an opposing group will ask the questions and stick bacteria or virus onto the membrane when questions are answered incorrectly. Students will be divided into groups of 6.

Each group will take turns as either group A or B. Each member of each group will take a role as one of the following See fig. I remain attached to the IgA until the enzyme cleaves me off o First SIgA — I bind to the bacteria and prevent them from binding to mucosal surface o Second SIgA- I bind the virus and prevent them from getting through the mucosa, multiplying and killing the cells o Enzyme- I cleave off the SIgA receptor so that the SIgA is released into the respiratory lumen.

Have one large piece of cloth or paper to represent the respiratory membrane. This should be taped onto the wall 3. There will be two opposing groups For larger class sizes, devide students into groups of six - each consisting of: a Two students to represent dimeric sIgA b 1 student to represent poly-Ig receptor.

The two pairs of students each pair holding hands to represent the dimeric nature of IgA representing Salivary IgA will then enter the class through the door while attached to the IgA receptor. The enzyme will follow behind. Each student will read their card to the class. Once inside the class, the student representing the enzyme will separate the receptor from the IgA pair.

The IgA pair will move to the membrane and stand guard. Group A will then be provided with 10 immune system questions to ask group B. Group B will have 1 minute to answer each question.

Every time a wrong answer is given, the card that contains that question is stuck onto the membrane behind the IgA and represents either a virus or bacteria. Six or more stuck cards represent an infection if more than half of them are bacteria or epithelial cell death if more than half are viruses. The groups will then take turns until each group has played the game as either group A or B 9.

The goal is to go through the activity with less than 6 cards stuck, getting more than 4 questions right. Classroom Discussion The following are sample questions to be given to students to use for discussion after the activity: 1.

What is the first line of defense that an invading pathogen must cross? How does saliva and its components protect against pathogens? Describe the structure of secretory IgA 4. Where is IgA made? To relate the activity to real life ask students why some individuals would have more virus and bacteria stick to the mucous membrane while others have less. Describe some diseases associated with low levels of IgA.

Please write out in short steps what has happened in the activity. When you are finished, take turns and describe these steps to a partner. Assessment: There are many possible modes of assessment for this activity. You may require that students write an individual essay describing what they acted out in class. The essay could include pictures to help support their work. Another assessment would be to have the students develop a poster, story, map, rap, song, poem, or representation of choice to indicate their knowledge of the topic.

Concept maps may be used as well to describe the process of IgA protection. Day 2 Simple dilutions Lab modified from Stanford University-sea urchin embryology Summary: Starting from a known concentration students will learn to use dilutions to determine the concentration of an unknown.

The students will acquire the following skills: 1. Making dilutions 2. Reading the meter on a simple home-made spectrophotometer as shown on pg 10 3. Keeping careful laboratory records 4. Graphing on linear graph paper 5. In this experiment, dilutions will be related to the real world.

Is it at the right concentration? When the ice melts- how much more dilute is the soft drink then? Using the soft drink as the material of interest, students will perform a simple linear dilution and construct a standard curve from which they will quantify unknown samples. For schools without a spectrophotometer, a simple one can be constructed using a light sensor, light source, filter and a meter. Soft drink absorbs light in the blue end of the spectrum.

Therefore a filter that primarily lets blue light through is used. Standard simple dilution of a soft drink. Unknowns should be made up ahead of time with student input. Students will obtain spectrophotometer readings of the dilutions and record the data in a table Demonstrate to the students the right way to read a spectrophotometer. If you are using a standard spectrophotometer, make sure it is on absorbance mode at nm.

Remember that the student standard curves may appear quite different from the one shown here. Soft drink simple dilution standard curve. Unknowns can be found by reading the K-Ohms or absorbance of the unknown and finding the concentration on the graph.

This lesson did a linear dilution. How would you do a serial dilution based on a factor of 2? In a subsequent lesson, secretory IgA standard protein will be diluted in a similar manner. What is the relationship between the K-Ohms readings and concentration?

How would you record the results for a solution that changes color with time? How much do you weigh? In grams? What would happen if the samples were not mixed well? How would this affect readings?

Assessment: Class participation and laboratory report. The antibody-conjugate binds to the SIgA in the standard or saliva samples. The amount of free antibody remaining is inversely proportional to the amount of SIgA present. After incubation and mixing, an equal solution from each tube is added in duplicate, to microtiter plate coated with human sIgA. The free or unbound antibody conjugate binds to the SIgA on the plate.

After incubation, unbound components are washed away. Bound conjugate is measured by the reaction of peroxidase enzyme on the substrate TMB.

This reaction produces a blue color. A yellow color is formed after stopping the reaction. Optical density is read on a standard plate reader at nm. The amount of peroxidase is inversely proportional to the amount of SIgA present in the sample4. Instruct students to: 1.

Rinse mouth with water 10 min prior to sample collection. Record time of day sample is collected 3. This can be done by adding 1 ml of water to the cryovial using a pipette. Students will work in pairs and the saliva from each pair will eventually be mixed. Students will use permanent markers to label their tubes with their two initials. Instruct students to imagine eating their favorite food and allow saliva to pool in the mouth.

With head tilted forward, student should drool down the straw and collect saliva into the cryovial It is normal for saliva to form. Repeat as often as necessary until 1 ml of saliva is collected, less the foam. Each group member will record the time he or she takes to collect 1mL of saliva. This will stimulate saliva production. The students will use the pipette to thoroughly mix the saliva while carefully avoiding foam formation.

The students will label the samples using their two last name initials one initial from each student in group, see the student instructions on page If pipettes are limiting, the teacher may carry out the transfer and mixing as a demonstration. Students will close the vials tight and bind the two vials with an elastic band.

When samples remain at room temperature longer than a few hours there is opportunity for bacterial growth that can invalidate the assay. While carrying the samples to the immunology lab for reading, place the saliva tubes in an ice box with ice. The rest of the steps will take place in an immunology laboratory as indicated on the protocol below. Usually during the homogenization process isotonic sucrose is added to the homogenization buffer to prevent osmotic rupture of organelle membranes since it is usually important to purify intact organelles.

Following homogenization, the homogenate is normally spun at low speed to remove any intact cells along with other large cellular debris. When this is done, the next step is subcellular fractionation.

Differential Centrifugation Differential centrifugation is one of the widely-used techniques to separate cellular organelles. A slight modification of this technic known as rate zonal centrifugation is also used frequently in which organelles, after a single spin, band in a tube according to their sedimentation rate. The technique of differential centrifugation is shown schematically in Figure 8. Size, density, and shape influence the movement of a subcellular particle in a centrifugal field.

This movement sedimentation results from the interaction between a particle's weight and the resistance it encounters in moving through a suspension medium and the relative centrifugal force exerted on the particle. Under a given centrifugal force, particles that are relatively large or dense will sediment more rapidly than particles that are smaller and lighter. With respect to the major components found in cells, the order of sedimentation is typically from most to least dense : nuclei, mitochondria, lysosomes, plasma membrane, endoplasmic reticulum, and contractile vacuoles.

Depending on the specific cell type, however, this order can vary. Additionally, differences in the rate of sedimentation are sometimes not large enough to provide separation of one organelle from another. Density-Equilibrium Centrifugation A second widely-used procedure for separating organelles is known as density-equilibrium centrifugation see Figure 8.

In this procedure, subcellular particles are layered on a density gradient and subjected to a very high centrifugal force. Usually, the density gradient is formed by layering increasing concentrations of sucrose solutions in a centrifuge tube. However, other solutions can be used, such as Percoll a colloid and cesium chloride. These latter two solutions, when spun, will spontaneously set up a density gradient, thus alleviating the need to as with sucrose manually layer sucrose solutions of varied density concentration.

During centrifugation, organelles initially layered on the density gradient will sediment until they arrive at the region of the gradient where the density of the suspension is equal to their own Figure 8.

At this point, an equilibrium condition is reached between the downward centrifugal force and the particle's tendency to float due to buoyancy, and sedimentation halts. Hence, this procedure is also known as isopycnic equilibrium centrifugation. Used alone, neither differential nor density-gradient centrifugation normally provides preparations containing organelles of sufficient purity. A common practice is to use both types of centrifugation procedures in sequence. However, even with this approach, obtaining desired organelles free from contaminants can require additional steps.

These steps can be many and varied, and often involve innovative approaches. Complete purification was finally achieved by feeding the cells Triton WR, a very low-density compound that preferentially accumulated into the lysosomes. This, in effect, decreased the density of the lysosomes, allowing them to be easily separated from the mitochondria using density centrifugation.

Figure 8. Schematic separation of organelles by differential centrifugation. See text for details. Organelle Isolation Figure 8. Schematic separation of organelles by density-equilibrium centrifugation. Marker Enzymes Isolation of any organelle requires a reliable test for the presence of the organelle. Typically, this is done by following the activity of an enzyme that is known to be localized exclusively in the target organelle. Such enzymes are known as marker enzymes.

For example, the enzyme acid phosphatase that cleaves terminal phosphate group from substrates and has a pH optimum in the acidic range is localized in lysosomes, while the enzyme succinate dehydrogenase is localized in mitochondria. By monitoring where each enzyme activity is found during a cell fractionation protocol, one can monitor the fractionation of lysosomes and mitochondria, respectively.

Marker enzymes also provide information on the biochemical purity of the fractionated organelles. The presence of unwanted marker enzyme activity in the preparation indicates the level of contamination by other organelles, while the degree of enrichment for the desired organelle is determined by the specific activity of the target marker enzyme.

Although marker enzymes reveal much concerning the purity of the organelle preparation, electron microscopy is generally used as a final step to assess the preparation's purity and the morphology of the isolated organelle. The Organism, Dictyostelium discoideum Dictyostelium discoideum is a eukaryotic microorganism popularly known as social ameba or slime mold. These individual cells will grow so long as a proper food source is available.

Under starvation conditions, however, to cells aggregate in an orderly manner to form a multicellular body which eventually differentiates into spores or stalk cells Figure 8.

Because of this unique life-cycle feature and the simplicity with which this organism is grown and handled, D. These include, but are not limited to, chemotaxis, signal transduction, pattern formation and differentiation, cell-cell communication, and endocytosis. Life cycle of Dictyostelium discoideum. See text, Appendix B, and Loomis for details.

Synopsis of the Experiment In this exercise, you will fractionate a homogenate of D. The purified fractions will be assessed using marker enzymes specific to each of these cell organelles. Aliquots of the homogenate will then be subjected to three different centrifugal forces and the pellets, as well as the supernatant fractions, will be assayed for marker enzymes specific to mitochondria, lysosomes, and contractile vacuoles.

These enzymes and their associated organelles are given in Table 8. The ratio of these three markers in various fractions will give an indication about the relative enrichment of one organelle over another in a given fraction.

The differential centrifugation steps used in the purification protocol make use of the fact that, in D. Table 8. Organelles and associated marker enzymes used in this exercise.

Students will therefore begin at step 4. Preparation of Homogenate 1. Resuspend in 1. Check homogenate under microscope for unlysed cells.

Repeat homogenization if necessary. Spin as in step 1 to remove unlysed cells and undispersed cell fragments. Discard pellet and save supernatant as homogenate. Centrifugation 4. Obtain 5 ml of diluted homogenate in a tube. Obtain four 4 microfuge tubes 1. Next, pipet 1 ml of the diluted homogenate into each of the four labelled microfuge tubes.

Keep microfuge tube A on ice; it is a control for total homogenate enzyme activity. Using Table 8. You will therefore need to perform three separate centrifugations. Before starting any run, check that the rotor contains a tube from each group in the class.

Also, make sure your tubes are in a balanced configuration in the rotor. Centrifugation protocol. Label three microfuge tubes Bs, Cs, and Ds.

Resuspend the pellets remaining in tubes B, C, and D in 0. Assay for Succinate Dehydrogenase The enzyme succinate dehydrogenase is an integral protein of the mitochondrial inner membrane. The major function of mitochondria is to generate energy ATP via oxidative phosphorylation. Succinate dehydrogenase, an FAD-containing enzyme, is involved in converting succinate to fumarate. In this assay, succinate is used as a substrate and nitroblue tetrazolium NBT as an artificial electron acceptor which changes to purple color when it accepts electrons.

Thus, the formation of purple color is directly proportional to enzyme activity. Pipet into each of these tubes: 0. Add 0. Note the starting time for each reaction. Stop the reaction by adding 2.

Read Absorbance at nm in a spectrophotometer adjusted to zero with the blank. Assay for Acid Phosphatase Acid phosphatase is present in lysosomes. The enzyme cleaves terminal phosphate groups and like other lysosomal enzymes operates maximally in acidic conditions. In this assay we will use a colorless compound, para-nitrophenol phosphate pNPP as the substrate for acid phosphatase. When the phosphate group of pNPP is cleaved, para-nitrophenol is generated.

Para-nitrophenol is a yellow compound that is easily measured in a spectrophotometer. Tabulation of Results Enter the data from your enzymes assays into Table 8.

In addition to completing this table, you will need to plot a bar chart; for each RCF-min, plot f for each marker. Tabulation of results. Notes for the Instructor This exercise was developed for a biology major junior-level undergraduate class.

Three hours time was sufficient when students worked in pairs. It is necessary that the students should have the outline at least 1 week in advance and that they study it before coming to the laboratory.

The exercise described here could be shortened or elaborated depending on available time and need. To elaborate the exercise, protein and other marker enzymes could be assayed. In addition, density centrifugation or another suitable step can be added for even better purification of organelles.

This exercise has been developed using an axenic Ax-3 strain of D. The same principle applies when fractionating organelles from other cell-types. However, conditions should be worked out for different cell-types. The clear advantage here is that contractile vacuoles shows clean separation from mitochondria and lysosomes as shown in Appendix C. Many cells which do not contain contractile vacuoles will not have such clear separation of organelles.

The enzyme assays described here should work well with other cell-types. I homogenize cells in a large volume up to 20 ml derived from ml culture for an entire class. I homogenize cells by passing them through polycarbonate filters see step 2 in the Laboratory Protocol and Appendix B.

Other methods of homogenization should also work well. However, it is important to use Buffer A because alkaline pH and a low ionic-strength buffer help dismantle cytoskeletons and therefore facilitates homogenization. See Das and Henderson for homogenization procedure using polycarbonate filters. Expected results and examples of assignment questions and answers are given in Appendix A. Instructions for growing and maintaining D. A table of the reagents that are required, instructions for the preparation of reagents, and the instruments, chemicals, and supplies that are required are given in Appendix C.

Additional points to remember include the following: 1. Keep all fractions on ice at all times. Organelle Isolation 2. Use of centrifuge: Tubes should be balanced, rotor lid should be tightly screwed on.

Laboratory assistant should run centrifuges. A microfuge could be used with the appropriate speed rpm equivalent to the desired RCF-min. Assay buffers can be added to the assay tubes well in advance while centrifugation is on. Add substrate to all tubes just before starting assay less than 10 minutes before assay.

Start reactions by adding enzyme solutions and stagger them as indicated below. It is imperative that each tube for the enzyme assay should be incubated for the indicated time.

This is generally achieved by staggering tubes by definite intervals 1 minute. This means start the reaction in one tube. After 1 minute start the reaction in the next tube and so on.