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Writing Competency Paper:

Murine Immune Response to Whole Cell Staphylococcus aureus

Student ID: 01558540

BIOS 442: Immunology

Dr. Matt Cruzen

February 24, 2017

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Murine Immune Response to Whole Cell Staphylococcus aureus

Introduction:

There are two main categories of immunity: innate and adaptive. Innate immunity is a response that is activated by the properties in which the antigen is made up of chemically. The skin, chemicals within the blood, and system cells make up the innate immunity. Adaptive immunity, also called acquired immunity, and can be defined as that which is obtained from the development of antibodies in response to antigen exposure.  In this report, feeder mice were exposed to a whole cell preparation of Staphylococcus aureus antigen and antibody production was quantified. Staphylococcus aureus is a bacterium found in the common normal flora of humans and has the ability to initiate both innate and adaptive immune responses due to the normal recurrence it displays in humans (Thammavongsa et al., 2015).  In mice, the time it typically takes to initiate an effective immune response is four weeks. This study investigates the early course of this response due to a limited time constraint. The results demonstrate how well a booster and non-booster mouse respond to antigen. Again, due to time restraints the antibodies produced by these mice are only from the initial primary immune response.

Two feeder mice were inoculated with formalin fixed Staphylococcus aureus. One additional control mouse was left non-immunized. After one week, one of the immunized mice was given a booster of formalin fixed antigen. This booster was given to sustain and improve the primary immune response elicited by the previous dose of the same antigen. Immune and nonimmune serum was collected for analysis via cardiac puncture. Antibody production in different mice varied due to antigen dosage. Based on previous immunization studies, a large amount of antibody in the two inoculated mice was to be expected (Prabhakara et al., 2011). Because the time frame within which the experiment was run did not allow for an effective secondary response, only the primary production of antibodies was expected to be observed. Additionally, if the specific class of immunoglobulin had been quantified in each of the inoculated mice at the beginning of the experiment, a higher titer of IgM (only present because initial response) would have been expected in the inoculated mice.

Methods: Treatment of Mice

The mice for the project were cared for consistently with food for the duration of the experiment. In order to differentiate between the four mice, physical alterations were made: mouse #1, the control, non-immune mouse, was left unchanged. Mouse #2, the immune mouse, had its right ear cut. Mouse #3, the boosted immune mouse, had its left ear cut. Mouse #4, the extra immune mouse, had both ears cut. Images of the four mice’s ear manipulations are shown below.

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Antigen preparation

The importance of antigen preparation is to ensure that the dosage prepared is appropriate to elicit a response and is not an overdose that will kill the mice. An overnight culture of 250ml Staphylococcus aureus  in Trypticase soy broth [TSB] was prepared by centrifuging at 6000rpm for 10 minutes in batches of 50ml in a Beckman Avanti centrifuge in 50ml conical tubes. The supernatant was removed, and the pellets were then re-suspended and pooled. The cells were then washed twice in phosphate buffered saline (PBS). After washing, the cells were fixed by adding 30ml of Safefix II (cat# 042-601 Fisher Scientific) overnight and incubated at room temperature on a rocking platform to ensure fixation of all cells. After fixation, antigen was washed three times with PBS and spun at 6000rpm for 10 minutes. The final pellet was then re-suspended in 30ml PBS and then quantified by Bradford assay and then stored at 4° C until use.

Antigen Quantification with Bradford Protein Assay

Antigen quantification via the Bradford assay is important in determining the concentration to use for inoculation. Using the reagent Coomassie Plus (Cat# 1856210) the assay was performed on the formalin fixed antigen (FFA). Serial 2x dilutions of a protein standard, bovine serum albumin [BSA] was assayed 10-3 to 10-6 in a 96-welled microtiter plate alongside serial 2x dilutions of the antigen, or serum. The linear range of assay for BSA (1mg/1ml stock) is 1.2 to 10.0µl/ml. The linear range of the assay performed as per product insert for BSA was .390625-3.125µg at 595nm. After the addition of reagent dye and incubation for 5 minutes, the A595 in the wells were measured on an ELISA plate reader.

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Figure 1: All experimental mice described in experiment.

Blue Circle: Mouse # 1, no ears cut, non-immune mouse.Yellow Circle: Mouse #2, right ear cut, immune mouse.

Black Circle: Mouse #3, left ear cut, booster mouse.Green Circle: Mouse #4, both ears cut, back up immune in case of escape.

ELISA Plate preparation

To begin, wells were coated with 100µl of antigen S. aureus at.5mg/1ml in each well. Plates were then washed 2x by discarding the well solution, 200μl of PBS (pH 7.4) per well were added and wait about two minutes and then repeated this cycle two more times. The wells were then blocked with 5% nonfat dry milk (5%NFDM) as prepared below in blocking buffer reparation. Once thoroughly filtered the NFDM blocking buffer, 100µl of the filtrate was added to each well and left overnight at room temperature. The following day the wells were washed 3x with PBS and the plates were left at room temperature until use.

Blocking buffer

The purpose of the blocking buffer in an ELISA is to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps (Thermo Fisher Scientific, 2017). A 50ml mixture of 5% non-fat dry (NFD) milk and PBS was created by adding 2.5 grams of NFD milk inside a conical 50 ml tube to make a solution of 20ml. The mixture was centrifuged and filtered through a whatman #1 filter.

Mouse inoculation

The protocol for the inoculation was to leave mouse #1 alone (control mouse with no ears cut), and to inoculate mouse #2 (right ear clipped), mouse #3 (left ear clipped), and mouse #4 (both ears clipped). A fixed amount of 75μl of the prepared S. aureus antigen at a concentration of 4μg/1ml was prepared in PBS. This solution was then injected IP into the nape of the neck of mouse #2, #3, and #4 on January 3, 2017.  The average weight of a 1-2-day old mouse is 20 grams, this measurement was taken into consideration when calculating the total volume of antigen produced. The calculated total volume of S. aureus antigen used in inoculation was 300μl. Same for booster shot for mouse #3 eight days after initial inoculation on January 11th, 2017.

Blood Serum collection and preparation

All blood was collected by cardiac puncture. Mouse #1 (control mouse) was bled on January 11th, 2017, and the following three mice (#2,3, and 4) were bled on January 17th, 2017.

Blood from immune and nonimmune mice were harvested by heart puncture. To prepare the mouse for the puncture, it was anesthetized using CHCl₃ (chloroform) inside a desiccation chamber for approximately two minutes. After anesthetization, the mouse was taken out and punctured with a 22 ½ gauge needle and blood was drawn into a 3ml syringe (figure 2). For easy access to the heart, the mouse was gripped by the scruff of its skin above the shoulders. The needle was inserted ~5mm from the center of the thorax towards the chin and inserted approximately 5-10mm deep at a 20° angle to the chest, vertically through the sternum, see figure 2.  Initial suction was created in the syringe, and the angle of the inserted needle was varied to promote blood flow into the syringe.   If insufficient blood was obtained, the mouse was cut open

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so the needle could be inserted directly into the heart, see figure 7. Depending on how much blood was collected, if there was an insufficient amount of blood still needed, the mouse’s spleen would be removed and soaked in 1mL of PBS in order to obtain enough antibodies to assay by ELISA. The blood samples were put into a centrifuge and spun at 8000rpm for 5 minutes to separate the serum from the blood clot. This is how enough serum was collected to assay by ELISA. The serum was then stored at 2-8°C until use. This procedure was done for mouse #1, #2, #3.

Figure 2: The needle is being inserted under the sternum to puncture the heart at an optimal angle for retrieval of blood.

Figure 3: Both mouse #1 and mouse #3 (shown) were cut open because insufficient blood was drawn from original extraction.ELISA (Enzyme-Linked Immuno-Sorbent Assay)

The prepared antigen plates were incubated with immune and nonimmune serum, followed by, anti-mouse conjugate (100µl, HRP conjugate, Cat #: 31491). Substrate of TMB was added for 5 minutes at room temperature before the 1M of HCL Stop Solution was added. Figure 4 illustrates what is happening in a single well in the ELISA plate test.

ELISA (Enzyme-Linked Immuno-Sorbent Assay)

Below is a visual representation of the of the assay used in the quantification of the antibodies specific to Staphylococcus aureus.   

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Figure 4: The purple circles represent the S. aureus and the orange squares represent the non-fat dry milk. The detection antibody represents the anti-mouse antibody (Horse Radish Peroxidase-Conjugate).

Antigen to Enzyme Fixation in ELISA Plate

In the preparation of the antigen to become fixed by our enzymatic soulution Figure 5 shows how the 96 well was set up for the procedure. The primary antibody of the mice is present in the serum dilution in the Pre-Bled, and Bled regions of Figure 5. In the fixation of the antigen

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Figure 5: The plate design of the ELISA plate used for quantification of mice antibodies.

Pre-Bled: In the non-immune pre-bled region, there was no antigen control at all present. Mouse #1 represent the pre-bled as the non-immune mouse.

Bled: In the bled region, you have a presence of antigen control present in the (B)-Booster mouse #3 and (I)- Immune mouse #2.

the second antibody conjugate is the anti-mouse antigen solution (HRP-Conjugate) prepared as an enzymatic linked solution. The substrate is the TMB which when added will convert our cell product a blue color upon contact with the prepared antigen in the cells. Once the stop solution (1M HCl) is added to the well, the solution will turn to yellow color verifying the reaction has taken place. The solution will be stocked by lowering the pH and the plate will be analyzed by the Bioassay spectrometer (Cat#: 0287-003), see figure 6.

Graph 1: Antigen vs. Absorbance at 595nm.

Immune and nonimmune serum was diluted in the Eppendorf  tubes with PBS at a ratio of 1:1000 (5µl serum to 1000µl  PBS). This was important in order to ensure the integrity of the samples contain the antigen at a concentration that is within the detection range of the antibody.

Table 1: Concentration of Absorbance in relational to Antigen (µg).

Bradford Protein Assay

Table 2: Absorbance of Antigen at 595nm on 1:64 serial dilution plate (multiple concentrations displayed).  The highlighted .3002 is the representative of Antigen Concentration after being read by spectrometer that correlate closest to the µg of antigen concentration between 6.25 and 3.125, which was found for our experiment to be 4µg/ml.

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Figure 6: After the addition of HCl (Stop Solution).

Dilutions Prepared:

Row A= 125µl of 5µl : 5ml, Serum : PBS, 1:1000, Dilution of Pre-Bled Serum. Row D, Column 1-6 = 200µl of 5µl : 5ml, Serum : PBS, 1:1000, Dilution of Bled

Immune Serum. Row D, Column 6-7 = 200µl of 5µl : 5ml, Serum : PBS, 1:1000, Dilution of Bled Booster

Serum.

125µl of 1:1000 diluted non-immune serum was added to rows A of the ELISA plate. 100 ml of PBS was then added to the wells in row B-C and columns 1-12. Using an eight-channel pipette, 25µl from A1 and A2...etc. were then added to B1 and B2...etc. and mixed. This was repeated through row C and after row C 25µl was disposed of in sink in order to 2x serially dilute the serum.  Next, 200µl of 1:1000 diluted serum from the immunized (mice #2) and booster (mice #3) mice was then added to row D, Column 1-6 (immune mice#2 serum dilution) and row D, Columns 7-12 (Booster mice #3 serum dilution). For each region on the ELISA plate in row D 100µl were moved and mixed from D1 and D2...etc. were then added to E1 and E2...etc. and mixed. This was also repeated for both regions through row H and then after row H 100µl was disposed of in sink in order to 4x serially dilute the serum.  

The sera were allowed to incubate in the ELISA plates with antigen for one hour (+/- one minute) at room temperature. After one hour, the plate was washed (3x) with PBS and smacked dried. All wells were then incubated with diluted 1:2500 conjugate (Promega, #w4021) for one hour (=/- one minute) at room temperature. After incubation, the plate was again washed with PBS (3x) and smacked dried. 100µl TMB substrate (Thermo Scientific, 34024) was then added to all wells until blue color developed. After ten minutes 100µl of stop solution (1M HCl)was added to each well which than reacted and turned yellow and was ready for Bioassay spectrometer analyzing at A485-200.

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Results:

Antigen Quantification with Bradford Protein Assay

Graph 2: Expanded View of the Standard Curve of Antigen vs. Absorbance at 595nm.

Blood Serum collection and preparation

The extracting of blood went well for mice #2 and 4 while having difficulties extracting blood from mice #1 and 3 resulted in puncturing through the heart based off human error. The compensation to puncturing through the heart and starting internal blood loss involved dissection of the living organism. The central trunk of the mice #1 and 3 were opened up exposing organs including the heart and much blood leaking everywhere. Using the syringe, there was several attempts to try and suck up as much blood left as possible before disposing of the mice. The results given error and not are:

Extracted 61.5µl of serum from mouse #1 (control mouse, no ears cut). Extracted 265µl of serum from mouse #2 (Right ear cut). Extracted 150µl of serum from mouse #3 (Left ear cut). Extracted 600µl of serum from mouse #4 (Both ears cut).

Thus, concluding a plethora of antibodies will be ready and present for analyzing via ELISA.

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ELISA (Enzyme-Linked Immuno-Sorbent Assay)

Discussion:

The results acquired in this experiment verified that both mouse 2 and 3 had elicited an immune response to the antigen Staphylococcus aureus. Furthermore, it was found that mouse 3 expressed more antibody production than mouse 2. As seen in the Elisa absorbance at 485-200, mouse 1 (control, non-immune Mouse #1) in the green on the chart, displays low values. This represents that there are not many antibodies present in the blood of the control. Values above .01 should be recognized as inconclusive data for mouse 1. Seen in mouse 2 (immune mouse; IgM) there is an increase in absorbance due to a correlation of exposure to antigen and then therefore production of antibodies. Similarly, mouse 3 (booster mouse; IgG) expressed far more due to double exposure of antigen. Any values higher than normal, for any respective category, are inconclusive. These are results of either bad pipetting or an unsuccessful blocking agent. Lower values for any respective category may be indicators of a bubble in the pipet, or the reagents did not yield enough positive results. This outcome was to be expected and aligned with the hypothesis. Because mouse 3 was given a booster shot 1 week into the incubation period, the immune response of mouse 3 reacted with an increase of antibody production. The booster shot provides a secondary antigenic exposure to the host and initiates a secondary immune response and the production of high affinity antibodies. Since the timeframe for this experiment was short, the data collect only reflected the very beginning of the secondary immune response for mouse 3, but was still enough to record a definite increase in antibodies compared to mouse 2.

In comparing the results found in Table 3 its essential to define the presence of antigen in the categories present. In the non-immune pre-bled region, there was no antigen control at all present. This points to the comparison of immune to non-immune samples in row A and D because the ELISA plate quantification (figure 5) required that both rows have the same 1:1000 dilution factor. The non-immune average overall was the lowest concentration of antigen at .1128 (row A, Column 1-6), while the immune had a substantial increase in concentration of antigen at .1815 (row D, Column 1-6). Overall though, the boosted mouse (mice #3) had the

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Table 3: ELISA Results from the absorbance measurements of serial dilutions.

The two circled average data points in the ELISA results demonstrates that the boosted mouse #3 had more antibodies present then the simple immune mouse #2.

largest marginal difference of antigen concentration being .2197 (row D, Column 7-12). These numbers justify themselves given the understanding that the non-immune antigen concentration was significantly lower based on the pre-bled (no antigen control) region over the bled (lots of antigen control) region. These comparisons are most reliable due to the same dilution in those set of wells on the ELISA plate and because no other correction factors were necessary for averaging the antigen concentration.

This information can be extrapolated to reflect what happens during an immune response not only in mice, but also in humans. Like the mice, human are also administered booster shots during times of high sickness susceptibility. This allows for a secondary and stronger immune response to take place. For example, tetanus shot boosters are often recommended every 10 years, after which memory cells specific against tetanus have lost their function or undergoing apoptosis (Mayo Clinic). Thus, the same importance for boosters in our mice in studying antibody production against S. aureus. Another thing to take into consideration is that in primary responses, the main antibody produced is IgM, where in a secondary response, it is IgG.  An administration of a booster shot increases the survivability of the host.

Based on the results, several inferences regarding immunity and immune system health can be made. The mice’s immune systems correctly recognized and defended against a foreign pathogen (S. aureus) that they had never encountered before. It was because of the diversity of the immune cells, mainly the B-cell, that allowed the mice to fight off the S. aureus bacteria. The diversity of the B-cells comes from the characteristic of class switching that allow for the vast majority of differentiation to occur. In total, there is about (10^18) different possibilities that a B-cell can arrange its antibodies’ light and heavy chains for the appropriate immune response (Murphy, 2012). Not only is the immune system adaptable, it is quick. Mice typically have an expressed and cleared response in about 4 weeks. However, it was seen that the boosted mouse began to express an increased response in less than 4 weeks.

Lastly, the results seen in the mice can be applied to humans as well. One could apply the same method of booster administration to a human and expect to see an increased secondary immune response. If the antigen could be quantified even more over time then there would be a greater abundance of IgG antibody. Given the longer amount of time allotted there would be a higher titer of IgG result in the mice.  It would be interesting to see how the abundance of antigen is affected given that one of the weapons S. aureus uses to evade the immune system is a protein called staphylococcal protein A (SpA) that turns the tables and neutralizes the antibodies first (Schneewind, 2015). Understanding how this protein may affect the substantial amount of antibodies produced to fight S. aureus over how many may be infected given a boosted mice would be important to analyzing the amount of antibodies it may take to fight a certain concentration of S. aureus.

For future experiments, things to change or take into consideration would be sample size, timeframe, and heart puncture practice. Given the smaller sample, errors can seem larger and out of proportion. With a larger sample of mice, there is more room for error. With a longer timeframe, the data reflecting a secondary immune response would be better defined. In this experiment, the data only shows a record of the very beginning of the secondary immune response. The heart puncture is probably the most important procedure in the entire lab. If done

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incorrectly, the subject’s blood becomes unusable and unable to analyze. Practicing the heart puncture procedure on backup mice or on fakes would provide the practice necessary to perform the heart puncture correctly and minimize human error.

The purpose of this experiment was to observe the immune response of mice to Staphylococcus aureus. The results show that a booster shot administered mouse produces more antibodies than that of a normal immune response mouse. It is because of the secondary immune response that a booster shot initiates that increases the expression of antibody producing genes. It shown that the immune response is both adaptable and fast acting as seen through the genetic variability of the antibodies of the mice.

Works Cited:

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"Block the Western Blot Membrane to Reduce Signal Background." Blocking Buffers forWestern Blot and ELISA | Thermo Fisher Scientific. Thermo Fisher Scientific, 2016.

Web. 17 Jan. 2017. <https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/blocking-buffers-western-blot-elisa.html>[accessed 2017 Jan 23]

Murphy, Kenneth. (2012) Variability of recombination chain. Janeway's Immunobiology, 8th edition. Garland Science. Figure 5.11

Prabhakara, R., Harro, J. M., Leid, J. G., Harris, M., & Shirtliff, M. E. (2011). Murine Immune Response to a Chronic Staphylococcus aureus Biofilm Infection. Infection and Immunity, 79(4), 1789–1796. <http://doi.org/10.1128/IAI.01386-10>[accessed 2017 Jan 23]

Schneewind, Olaf. "An Antibody to Beat Staph at Its Own Game." Science Life. The University of Chicago Medicine & Biological Sciences, 28 Jan. 2015. <https://sciencelife.uchospitals.edu/2015/01/19/an-antibody-to-beat-staph-at-its-own-game/>[accessed 2017 Jan 23]

Tetanus: Prevention, Mayo Clinic, 2006-09-21, archived from the original on 2008-06-24, retrieved 2008-07-17

Thammavongsa, V., Kim, H. K., Missiakas, D., & Schneewind, O. (2015). Staphylococcal manipulation of host immune responses. Nature Reviews. Microbiology, 13(9), 529–543. <http://doi.org/10.1038/nrmicro3521>[accessed 2017 Jan 23]

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