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The Immune System Add in library

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Question:

Discuss that  immune system protects the body from various harmful substances and various infectious agents by various molecules and cells?

 

Answer:

Introduction:

The immune system protects the body from various harmful substances and various infectious agents by various molecules and cells. The immune system has the ability to tolerate the presence of indigenous substances of the body and it prevents foreign substance entering into the body. The immune system is capable developing response against the foreign substance that has entered into the body such as bacteria, virus, parasites and toxins that are surrounded by antigens. This discriminatory ability of the immune system provides protection from infectious diseases, as most of the associated toxins or agents are recognized as foreign substances by the immune system (De Ranieri, 2013). The immune system of the body depends upon the functions three types of white blood cells (WBC) that are obtained from bone marrow:

  • Phagocyte cells - The dendrite cells and macrophages are the types of phagocyte cells that are present in tissues and blood waiting to engulf foreign particles.
     
  • T cells – Some of the White blood cells leaves the bone marrow and reach the thymus gland and differentiated into thymus lymphocytes or T cells.
     
  • B cells – The White blood cells that do not reach the thymus gland becomes the B cells or the B lymphocytes.

There are mainly two types of immunity in the body; they are passive immunity and active immunity. Passive immunity is the type of immunity in the body in which antibodies are injected in the body. It is called passive immunity as the antibodies are not created but transfused pre-formed antibodies. The examples of passive immunity are maternal antibodies that are obtained from breast milk or trans-placental and transfused antibodies such as varicella, rabies, tetanus immune globulins. The active immunity is created in the body when a foreign substance enters into the body and the immune system recognizes it and produces particular antibodies, this is called active response. The active can be further divided into two subcategories, innate immunity that is the protective immunity system with which a person is born and adaptive immunity that is humoral immunity and cell mediated immunity. The polyclonal antibodies create multiple B cells to target a particular epitope antigen. However, the monoclonal antibodies create single B lymphocyte for a particular epitope antigen (Galan, et al., 2009).

The clinical diagnosis of an infectious disease shows the interaction between the micro organism and the host. Some of symptoms of infectious disease are clear enough to be recognized clinically. However, various pathogens create varied range of symptoms in the host. Thus, it is essential to identify the specific type of etiologic agent by using microbiologic laboratory methods. The etiologic agents of disease are identified by the principles of Diagnostic medical microbiology. The functions of the clinical microbiology laboratory involve testing specimens from the patients for micro organisms and determine the cause of illness and provide specific information regarding the in vitro function of the antimicrobial drugs against the recognized micro organism (Guicai, et al., 2008).

 

Immunological tests are performed for an antigen to recognize antibodies to its pathogen or by the use of antibody; the antigen of the pathogen is detected through the specimen of the patients. The enzyme immunoassay uses the antibodies connected to the enzymes to recognize antigens and quantify antibodies. The Enzyme linked immuno sorbent assay (ELISA) test is sensitive to most of the enzyme of immunoassay and are therefore, used for screening.

The fibrinogen is a type of glycoprotein present in the human body and helps in the formation of blood clots. It is a soluble, complex glycoprotein, plasma glycoprotein and large substance. The fibrinogen is converted by thrombin to fibrin during the formation of blood clot. The gliadin is type of protein present in the genus Triticum such as wheat and other cereals (Gujral, et. al., 2012). Gliadins are the components of gluten. The following enzyme linked immuno sorbent assay (ELISA) test is performed to determine the concentration of the proteins human fibrinogen and gliadin. This particular experiment is conduct to know the quantity of human fibrinogen present in the human body as the fibrinogen helps in blood clotting and its presence is very much essential in the human blood. The gliadin is a type of protein present in the wheat, it is the soluble component of gluten and the human body is intolerant to it and causes the celiac disease in the small intestine. The gliadin generally damages the lining of the small intestine and the villi get flattened that normally disables the intestine surface to absorb food, and perform it regular functions. However, there is no cure for the celiac disease; therefore, the individuals suffering from this disease would be advised to avoid gluten free diet in their entire life. The gluten free diet is generally not possible to get. Thus, ELISA test is essential to determine whether the particular sample of food contains gliadin residues (Kaukinen, et al., 2007).

The principles of ELISA test are as follows:

  • The test includes sensitive immunoassay in which enzyme is linked to the antigen or antibody as a mark for detection of the particular protein, specially the antibody or antigen.
     
  • ELISA includes detection of analyte in a liquid sample using a dry strips or liquid reagent.
     
  • In dry analysis, reflectometry can be used to read strips. The reading is basically dependent upon detection of intensity of transmitted light with particular wavelength by the spectrophotometry.
     
  • The amplification of signal during the analytic reaction is the factor on which sensitivity of detection is depended.
     
  • There are two types of variation in the methods of ELISA – (1) it is used to recognize the presence of antigens which are previously detected by the antibodies. (2) It is used to determine antibodies that detect the antigens (Lupo, et al., 2013).

Aim:

The aim of this particular experiment is to establish two standard curves by ELISA test from the samples containing known concentrations of the human fibrinogen protein and gliadin protein. The ELISA test was used in this experiment as it is sensitive to most of the enzyme of immunoassay and are therefore, used for screening.

Hypothesis:

The human fibrinogen has a dual function; the first is to act on a cofactor in platelet aggregation and other to yield monomers that polymerize into fibrin. The gliadin is a component of gluten and is present in most of the bakery products. However, it is responsible for disease like celiac disease and allergies such as Baker’s asthma.

 

Materials required:

For the human fibrinogen experiment – Streptavidin Peroxidase Conjugate (SP Conjugate), diluents N Concentrate, Wash Buffer Concentrate, Biotinylated Human Fibrinogen, Chromogen Substrate, Fibrinogen Micro plate, Fibrinogen Standard, Sealing Tapes and Stop solution. Precision pipette for dispensing and making dilutions, test tubes, microtiter aspirator, distilled H2O, microtiter plate reader, assorted glassware for the preparation of buffer solutions and reagents, timer, centrifuge for collection of sample and anticoagulant for the collection of specimen. Assay plate, HRP conjugate, wash buffer, stop solution. Micro plate reader that has the ability of measuring absorbance at 450 nm should be provided and that has correction wavelength at 570nm. Manifold dispenser and squirt bottle.

Discussion:

Test for Human Fibrinogen - The Fibrinogen test apparatus was highly sensitive, the two site enzyme linked immuno assay (ELISA) for calculating the Fibrinogen in biological specimens of humans. The soluble fibrinogen circulates in the body and produces substances like insoluble fibrin clot that is needed during blood coagulation. Fibrinogen is a unique reactant that is useful marker for inflammation and infection. The ELISA apparatus therefore, can be used to calculate the fibrinogen in biological specimens. In this experiment the fibrinogen present in the specimen sample reacts with the anti fibrinogen antibodies that adsorbed in the surface of polystyrene microtiter wells. The unbound proteins was washed away, the anti fibrinogen conjugate with horse radish peroxidise (HRP) was added. The enzyme labelled antibodies binds with previously complexes of fibrinogen. Another washing steps was followed, the enzyme bound with the immuno sorbent was assayed after addition of chromogenic susbstance like 3,3’,5,5’-tetramethylebenzidine (TMB). The concentration of fibrinogen specimen varies with the quantity of bound enzyme (Mothes, et al., 2009). Therefore, the absorbance was measured of the fibrinogen concentration in the test specimen. Fibrinogen quantity in the experiment specimen could be interpolated from the standard curve formed from the standards and checked in the sample dilution.

The principle of the ELISA experiment involves the competitiveness of inhibition enzymes during immuno assay technique. The specific antibody of fibrinogen should be pre coated in the micro plate. Specimens should be added efficiently in the microtiter plate wells with human fibrinogen and HRP conjugate. The competitive inhibition reaction should be launched between human fibrinogen and HRP conjugated fibrinogen with the particular fibrinogen pre coated with antibodies. However, if high amount of human fibrinogen specimens are present then low amount of antibody bounded with HRP conjugate fibrinogen should be added. The substrate solution should be added carefully with the respective wells. The colour would simultaneously develop opposite to the amount of human fibrinogen specimen (Piva and Plebani, 2010). When the colour development is stopped during the experiment, the intensity of the colour should be measured. The minimum detected level of human fibrinogen is generally less than 25 ng/ml. The sensitivity of the experiment or the Lower Limit of Detection (LLD) is known as the lowest protein concentration that could be separated from zero.

The limitation of the experiment was like reproducible and reliable outcomes that are gained when the experiment is carried out through complete understanding of the guidelines within the instruction and good laboratory practises. The factors that may affect the functions of the experiments involves cleanliness of the glassware, accuracy of the reagent, washing technique, proper distilled water, accuracy of sample pipettes, incubation temperature or time. The substitute reagents should not be mixed with other resources. The experimental apparatus that is used should not have crossed the expiry date, however, if the specimen provides higher values than the normal standard, the specimen should be diluted and the experiment should be repeated again, and the binding can be influenced by the defects in operator, washing techniques, pipettes techniques, incubation temperature or time and the validity of the apparatus used in the experiment (Salim, et al., 2007).

 

The technical hints for performing the experiments are while mixing the protein solution, foaming should always be avoided, the cross contamination should be avoided and in that case pipette tips should be changed during addition of each standard level, between reagent additions and between sample additions and also the different reservoirs should be used for each reagents. The experiment could be improved by using an automated washer plate, rotating the plate 180 degree during washing steps, providing a 30 sec soak time between additions of wash buffer. In order to get accurate results, adhesion of plate sealers in the time of incubation is essential. Until the substrate solution is added to the plate it should be colourless. The substrate solution should be protected from the sunlight. The substrate solution should gradually change from colourless into blue gradation. The stop solution should be added in the plate in similar way as the substrate solution was added (Tonutti, et al., 2009). Upon the addition of the stop solution the colour of the solution will gradually change from blue to yellow colour. However, in case some wells are green in colour, this indicates that the stop solution was not properly mixed with the substrate solution.

While storage of the specimen, the following points should be maintained, the apparatus should be kept unopened and stored at the temperature at 2 – 8 degree centigrade and the entire kit should be kept in a sealed bag and minimize the exposure to damp air. As the apparatus would be used throughout the experiment, the expiry date of the apparatus should be checked before starting the experiment. The opened test apparatus should be remain stable and checked whether no external source disturbs it. The absorbance measurement would be effectively taken if the microtiter plate reader is with a bandwidth of 10 nm or less than it and the optical density range should be within 0 – 3 OD or more than 450 nm wavelength is preferable (Vida, et al., 2012).

The sample collected and stored were serum, plasma and urine. The serum separator tube should be used and time should be provided for the sample to clot for 30 minutes before its centrifugation for 15 minutes at 1000 g. The plasma was collected through EDTA, heparin or citrate as anticoagulant. And then centrifuge for 15 minutes at 1000 g within 30 minutes of its collection. After assay of the sample immediately store in -20 degree centigrade. The urine sample was collected using sterile collection tube and any external particles was removed through assay and immediately stored in -20 degree centigrade (Vida, et al., 2012).

However, though the human fibrinogen ELISA test, the medical science would be helpful to determine the level of fibrinogen amount present in the human blood and if any abnormalities is seen during the blood coagulation of the patient, this experiment would be very effective in case of determining the level of fibrinogen present in that patient, as the fibrinogen in the human body is responsible for blood coagulation. The scientist could also locate the human fibrinogen components artificially and develop artificial fibrinogen capsules to the patients. 

Test for Gliadin – For the quantitative analysis, a micro plate or strip is required for the experiment. The experiment should be performed at 22 – 23 degree centigrade, and the experiment is preferable to conduct in the laboratory incubator in order to maintain the stable conditions. The principle of the gliadin ELISA experiment is that when the test sample would be added and if gliadin residue is present it would bind with the respective antibodies, the enzyme labelled conjugate was added and it would bind with the gliadin residue and when the substrate would be added that was converted in the presence of enzyme conjugate to provide blue colour if the particular sample contains gliadin residues. However, a yellow colour will also form when acid would be added to the stop solution (Piva and Plebani, 2010).

 

In order to get quantitative analysis, the results must be taken on micro plate or strip reader. This would also help while determining gluten free status, based on the standard of cut off value. While in qualitative testing the results must be taken visually depending upon the colour of the standards.

The gliadin substance is intolerant to some human beings as it affects the lining of the small intestine and flattens the villi; thus, the function of the intestine is highly affected by the gliadin residue in the body, this disease is called celiac disease. The patients suffering from this disease is advised to have gliadin free diet throughout their entire life. However, gliadin free products are generally not possible to get in the market. Therefore, the ELISA test for measuring the level of gliadin present in the food substance.

The advantages of ELISA test are as that is convenient and quick, the unknown antigen of unknown concentration could be detected as in this experiment the antibody binds with the respective antigen. The procedure of the experiment is generally safe and do not involve radioactive substance and requires dilute sulphuric acid. This test is widely used in various experiments. As compared to other experiments the ELISA tests has many advantages and are very accurate. The experiment is highly sensitive, exact and favourable with other methods like radio immune assay (RIA) tests (Piva and Plebani, 2010).

The disadvantages of the ELISA test are that in the experiment only monoclonal antibodies could be used in this experiment. Polyclonal antibodies cost less than monoclonal antibodies, it is difficult to find out monoclonal antibodies, if the blocking solutions are ineffective the negative controls might show positive results and also could bind with open sites in the well. The terms like enzyme or substrate reaction are very short term and therefore, micro wells should be used in that place (Gujral, et. al., 2012).

The ELISA test is done in three different ways, the indirect test, direct test and the sandwich test. The indirect ELISA test is conventional but efficient, in this experiment there are two steps, first steps involves binding with the primary antibodies and then binding with the secondary antibodies. The primary antibodies are first of all incubated with the antigen in the incubator and then secondary antibodies are added to it. However, this process may include cross reaction with the non specific signals. The direct ELISA is time saving and simple. At first in the direct test, the antigen is adsorbed in a plastic place and then introduced to another protein so as to block other binding sites. The enzyme antibody complex is provided to adsorb to antigen in a separate reaction. The enzyme antibody bound antigen is left out after the excess antibody complex is washed off. After the addition of enzyme substrate, the enzyme is recognized indicating the sign of antigen. The sandwich ELISA test is highly sensitive (Piva and Plebani, 2010). This test is very uncommon to various ELISA tests; however, it is highly preferable in getting sample antigen detection. Many commercial ELISA test are based upon sandwich ELISA test as it provides efficient and appropriate results. The sandwich ELISA test indentifies antigens in two layers of antibodies that are detection and capture. Thus, is can be concluded that the various ELISA test are effective enough to detect the amount of specific substance present in the given sample through antigen antibody complexes (Gujral, et. al., 2012)

 

References:

De Ranieri, E. (2013). Spin caloritronics: Put to the test. Nature Nanotech.

Galan, X., Hoyos, M., Cacho, E., Goirigolzarri, I. and Munujos, P. (2009). Analytical Performance of a New Gliadin-Activated Anti-tTransglutaminase ELISA Test. TOAUTOJ, 1(1), pp.79-85.

Guicai, L., Xiaoli, S., Ping, Y., Ansha, Z. and Nan, H. (2008). Investigation of fibrinogen adsorption on solid surface by quartz crystal microbalance with dissipation(QCM-D) and ELISA. Solid State Ionics, 179(21-26), pp.932-935.

Gujral, N., Suresh, M. and Sunwoo, H. (2012). QUANTITATIVE DOUBLE ANTIBODY SANDWICH ELISA FOR THE DETERMINATION OF GLIADIN. Journal of Immunoassay and Immunochemistry, 33(4), pp.339-351.

Kaukinen, K., Collin, P., Laurila, K., Kaartinen, T., Partanen, J. and Mäki, M. (2007). Resurrection of gliadin antibodies in coeliac disease. Deamidated gliadin peptide antibody test provides additional diagnostic benefit. Scand J Gastroenterol, 42(12), pp.1428-1433.

Lupo, A., Roebuck, C., Walsh, A., Mozola, M. and Abouzied, M. (2013). Validation Study of the Veratox R5 Rapid ELISA for Detection of Gliadin. J AOAC Int, 96(1), pp.121-132.

Mothes, T., Prause, C., Probst, C., Schlumberger, W., Hauer, A., Koletzko, S., Laass, M., Richter, T., Stern, M., Uhlig, H. and Zimmer, K. (2009). W1233 Serodianosis of Coeliac Disease Using a Newly Developed ELISA for Antibodies Against Deamidated Gliadin (Anti-Gliadin (GAF-3X)-ELISA). Gastroenterology, 136(5), p.A-683.

Piva, E. and Plebani, M. (2010). The Esr Test: An Old Test With New Contents. Journal of Medical Biochemistry, 29(4).

Piva, E. and Plebani, M. (2010). The Esr Test: An Old Test With New Contents. Journal of Medical Biochemistry, 29(4).

Salim, M., O'Sullivan, B., McArthur, S. and Wright, P. (2007). Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA. Lab Chip, 7(1), pp.64-70.

Tonutti, E., Visentini, D., Picierno, A., Bizzaro, N., Villalta, D., Tozzoli, R., Kodermaz, G., Carroccio, A., Iacono, G., Teresi, S., Chiusa, S. and Brusca, I. (2009). Diagnostic efficacy of the ELISA test for the detection of deamidated anti-gliadin peptide antibodies in the diagnosis and monitoring of celiac disease. J. Clin. Lab. Anal., 23(3), pp.165-171.

Vida, V., Padalino, M., Barzon, E. and Stellin, G. (2012). Efficacy of Fibrinogen/Thrombin-Coated Equine Collagen Patch in Controlling Lymphatic Leaks. Journal of Cardiac Surgery, 27(4), pp.441-442..

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