Testing of Functional Capacity of SRBC Serum Complement Components - Experiment Results and Discussi

What Human Serum Sample Was Labelled x? How Did You Come To This Conclusion?

What Human Serum Sample Was Labelled y? How Did You Come To This Conclusion?

Why Were The Erythrocytes Coated With Antibody?

Why Was Yeast Used In One Of The Human Serum Samples?

How Would You Plot The Above Results As a Graph?

What Other Controls Could You Incorporate In The Experiment?

Are The Results What You Expected? Explain?

The complement system has been defined as the group of proteins which activated in order to perform cellular lysis. This system has been known as a complement cascade which is found as a part of the immune system which enhances the ability of the phagocytic cells and the antibodies to kill the microbes and remove the damaged cells from an organism (Meri 2016). Two of the main features associated with the complement system is the promotion of inflammation and an attack on the pathogen’s cell membrane (Jin and He 2017). This process is performed by the formation of the membrane attack complex. This experiment will test the functional capacity of (SRBC) serum complement components which helps to lyse the red blood cells of sheep which are pre-coated with rabbit anti-sheep red blood cell antibody known as haemolysin. The classical complement pathways has been found to be activated when the SRBC coated with antibody has been found to be incubated with the test serum (Nayak, Portugal and Zilberg 2018). This report will discuss the aim of the experiment at first followed by the procedure used to perform the experiment to get the results. Finally, the paper will end with a discussion of the results by a conclusion.

This experiment will aim at mixing up of two serum samples (Y and X) in the laboratory to understand their origin. In order to determine the origin of these antigens, the amount of complement consumed must be calculated. This amount will be calculated by the preparation of antibody-coated sheep erythrocyte in order to determine the residual complement activity. After ending the experiment, it can be stated that the determination of the sample origins will be clear.

1. 150 microliters of human serum either labelled as X or Y.
2. 150 microliters of human serum with pre-incubated yeast at 37 degrees C for 30 minutes either labelled as X or Y. 3.5 mL of CFD (complement fixation diluent)- 5X solution.

4. 0.2 mL of SRBC (Sheep Red Blood Cells)
5. 20 microliter of Rabbit anti-sheep cell haemolysin solution
6. 96 well plate
7. Parafilm
8. Ice
9. 2.5 mL tubes

1. 1 mL of 1X haemolysin solution is prepared by diluting the same to 1:50 with the help of 1X CFD buffer.
2. 0.2 mL of SRBC was prepared by adding 0.3 mL of 1X CFD by gentle inversion mixing process.
3. The same was centrifuged at 600g x 5 minutes.
4. The supernatant was discarded and the cells were washed for another two times.
5. After performing the final wash, the cells were centrifuged at 900g x 5 minutes in order to pack the cells.
6. After this process, the supernatant was discarded and the cells were resuspended in 1X CFD to prepare a 10% solution. This solution amounts to 0.1 mL of packed cells which are further resuspended in 1 mL total CFD buffer.
7. A dropwise solution of haemolysin was added to the cells by constant stirring inside a water bath.
8. The same was incubated in a water bath for 30 minutes at 30-degree centigrade.
9. Gentle mixture of cells after 15 minutes was performed.
10. 0.1 mL sheep erythrocytes were removed and added to 1.9 mL 1X CFD in order to make a 1% sheep erythrocyte solution.

1. A 96 round-bottomed well microtitre plate was labelled and 100 microliters of complement fixation diluent were added to each of the first three rows of 12 wells (A, B and C). The first two wells of row D (control samples) were also loaded in the same way as the other wells stated before.
2. 100 microliters were transferred from the Human serum X tube into the well of row A of the microliter plate. 1X CFD was mixed and 100 microliters was removed from well 1 and transferred to well 2. Again, CFD was mixed in well 2 and then 100 microliters were removed from it and transferred to well 3. This process is known as a doubling serial dilution procedure and was carried out till well 12. After the mixing was complete and 100 microliters were discarded from well 12 into a disinfectant jar.
3. The same procedure was repeated for human serum Y to row B of the microtitre plate.  
4. The same procedure was repeated using 1X CFD to row C of the microtitre plate.
5. Antibody coated sheep erythrocytes was mixed thoroughly by inverting the tube several times. Then, 50 microliter EA has added all the wells present in rows A, B and C of the microtitre plates.
6. In the first two wells of row D, 50 microliter 1X CFD was added.

The plate layout has been shown below:

7. The well contents were mixed carefully by tapping the plate and finally, the plates were covered with parafilm and finally incubated at 37-degree centigrade for 30 minutes and then it was mixed carefully after 15 minutes.
8. After 30 minutes, the plates will be looked for EA cell lysis. However, it can be stated that if the SRBC has not settled, the plates must be left at 4 degrees C to let the cells settle before reading the final results.
9. After setting, the amount of hemolysis will be observed and compared with the size of the EA plate in each of the experimental wells as a percentage to the control wells.  

The picture below shows the condition of the cells in the pate wells:

Fig 1: Microtitre plate observed after the experiment.

The tables below show the results:

Fill in your tables using the layout below as a guide.

Methods

Table 1:  % lysis row A of the microtitre plate

Table 2:  % lysis row B of the microtitre plate

Table 3:  % lysis row C of the microtitre plate

Table 4.  % lysis row D of the microtitre plate.

Fig 2: Graph showing no points in the coordinate because of 0 value of results

 Thus, from the above results and graphical analysis, it can be stated that the results were negative. The wells did not show any percentage lysis which proves the fact that the complement pathway was not activated. Human serum samples which were incubated with Yeast cells, failed to activate the complement system because the yeast might have been degraded before the experiment was performed (Trendelenburg et al. 2018). Another factor which led to the arousal of a negative result was improper concentrations of every addition which was done in this experiment (Mishyna et al. 2018). The incubation times may not have been perfect which did not have enough times to the components to mix properly. SRBC settling time may not have been enough for the reaction to occur properly and thus the complement system failed to get activated which could have resulted in cellular lysis (Mamidi, Höne and Kirschfink 2017). However, if this was not the case, then the classical complement pathway may have been activated and the cells might have been lysed. Inflammation would have been found to occur in the cells giving rise to the formation of membrane attack complex and the cells would have been lysed if there was an observed percentage of lysis in the results (Salvador-Morales and Sim 2016). This is the overall brief discussion which can be done based on the negative results of the experiment.

Conclusion

On a concluding note, it can be stated that the experiment shows that the complement pathway is not activated according to the process stated in the introduction. There is no formation of membrane attack complex or inflammation resulting in the degradation of cell walls which would have caused the cellular lysis. Negative results could have been prevented by considering the precautions necessary to perform a complement assay in a microtitre well. None of the wells shows lysis which could have occurred due to expired materials or the use of wrong concentrations. Precautions should be taken to prevent a negative result from the next time while performing these types of experiments.

References

Jin, J. and He, S., 2017. The complement system is also important in immunogenic cell death. Nature Reviews Immunology, 17(2), p.143.

Mamidi, S., Höne, S. and Kirschfink, M., 2017. The complement system in cancer: ambivalence between tumour destruction and promotion. Immunobiology, 222(1), pp.45-54.

Meri, S., 2016. Self‐nonself discrimination by the complement system. FEBS letters, 590(15), pp.2418-2434.

Mishyna, M.M., Mozgova, Y.A., Kovalenko, N.I. and Zamaziy, T.M., 2018. Standard protocols to laboratory classes in microbiology, virology and immunology for the II year English media students of medical and dentistry faculty (Part 1).

Nayak, S., Portugal, I. and Zilberg, D., 2018. Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells. Veterinary immunology and immunopathology, 199, pp.39-42.

Salvador-Morales, C. and Sim, R.B., 2016. Complement activation. In HANDBOOK OF IMMUNOLOGICAL PROPERTIES OF ENGINEERED NANOMATERIALS: Volume 2: Haematocompatibility of Engineered Nanomaterials (pp. 303-330).

Trendelenburg, M., Stallone, F., Pershyna, K., Eisenhut, T., Twerenbold, R., Wildi, K., Dubler, D., Schirmbeck, L., Puelacher, C., Rubini Gimenez, M. and Sabti, Z., 2018. Complement activation products in acute heart failure: potential role in pathophysiology, responses to treatment and impacts on long-term survival. European Heart Journal: Acute Cardiovascular Care, 7(4), pp.348-357.