Lung cancer is a cancer that starts from the lung. There are three different types of lung cancer. Such as- non-small cell lung cancer, small cell lung cancer and lung carcinoid tumor. Compare to other cancer, lung cancer, it’s difficult to detect lung cancer in its earliest stage because most people won't have any symptoms. Once symptoms develop, the cancer is usually advanced and no longer curable.
Even for diagnose a chest x-ray won’t show the early-stage of lung cancer. The majority of people have no symptoms until their lung cancer is at an advanced stage. Lung cancer is the leading cause of cancer death all over the world. The alternative way the lung cancer can be detected at early stage by biomarker. (N. LynnHenry, et al, 2012)
Biomarker is a biological marker. It is actually measureable indicator of some biological state or condition. A biomarker means biological molecule usually found in tissue, blood or other type of fluids that can give sign of normal or abnormal process or condition or disease such as cancer.
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There are variety of biomarker can be used, such as protein, nucleic acid, antibodies, peptides and so on. For lung cancer lots of protein is used for biomarker. Most of the proteins are not sensitive enough to detect the early stage of the lung cancer. But recent research study says CKAP4 protein can be a potential biomarker , which is sensitive enough to detect the lung cancer at early stage among all the proteins.( Kengo Yanagita, et al, 2018)
By using CKAP4 protein can easily produce polyclonal antibodies in laboratory animals such as rats, mouse, rabbits, chicken, horse, sheep and so on. (Marlies Leenaars, et al, 2005). Rabbits is usually use for production of polyclonal antibody due to it’s convenient size, ease of breeding, long life p and for more antibodies production. ( Stills 1994 ). The production of polyclonal antibodies process involve with a five-steps that starts with preparing the antigen.( Saylor,et al,2009).
Following this, an adjuvant is chosen and prepared to act and respond to the antigen. The step three is the selection animal. A solution containing both the antigen and adjuvant is to be injected into the animals. These injections continue over a set period until the animal has produced the desired antibody. The final step is that taking out the blood from the animal for further assessment .
After extracting the blood from the animal, the blood contains the antigen-specific polyclonal antibody. There are two ways to check the specificity of the protein. To able to test the specificity, ELISA would be a great way. Because it is easy to perform, highly specific, give quick and accurate rest. ( Sblattero, D. , et al, 2000)
Antibodies, are known as immunoglobulins. It is secreted by the B cells (Plasma cells) to neutralize antigens like bacteria and viruses. The shape of the antibodies are Y, which contains four polypeptides- two heavy chains and two light chains. The shape ‘Y’ contains antigen binding site which is very specific for particular epitope on an antigen. The antibodies are classified into two types such as polyclonal antibodies and monoclonal antibodies.
A polyclonal antibodies are basically collection of antibodies which are derived from various B cells (Plasma cells). they have the ability to recognize and bind to multiple different epitopes of a single antigen. The advantages of polyclonal antibodies is higher than monoclonal antibodies due to its binding specificity to antigen. Due to its binding ability, it is largely use in the medical sector and life science sector for medical treatment. (Claire, et al, 2006)
Polyclonal antibodies characteristics:
Polyclonal antibodies (PAb) has lots advantages due to recognize multiple epitopes on the same antigen. Polyclonal antibodies are able to recognize multiple sites on an antigen. The serum obtained contains a number of different antibodies of different affinity. Comparatively, the production and extraction of polyclonal antibodies is inexpensive, they require minimal production technology minimal training needs to use the technology, consequently, lower laboratory costs.( Neil S. Lipman, et al, 2005).
The production of polyclonal antibodies is very quick. The purified antibody ready to use in under four months. It is highly stable and tolerant to pH or buffer changes and east to store. It is deal as the capture antibody in a Sandwich ELISA. Greater ability to quickly capture the target protein. It offers greater sensitivity for detecting proteins that are present in low quantities in a sample since multiple antibodies will bind to multiple epitopes on the proteins.
Production of polyclonal antibodies:
Antibodies are basically used for research and diagnostic purposes are often obtained by injecting a lab animals. There are some critical steps involved in production of polyclonal antibody. The first step is the preparation of the antigen. Because , before injecting the antigen into the animal, the must go through quality control whether it is purified or not. Due to purification result, it increased the number of antibodies. ( Leenaars et al. 1997 ).
The second step is the selection of animal speicies. The most frequently used animal species for PAb induction in the laboratory setting are rabbit,goat, mouse, rat, sheep, horse, guinea pigs,hamster. (Marlies Leenaars, et al, 2005).For the production of PAbs, rabbits are used most often because of their convenient size, ease of handling and bleeding, relatively long life p, and adequate production of high-titer, high-affinity, precipitating antiserum ( Stills 1994 ).
When larger amounts of PAbs are needed, farm animals can be used such as sheep, goats, and horses. In some cases requiring large amounts of PAbs, chickens may be used ( Erhard et al. 2000 ; Schade et al. 1996 ). Recommended age of animals for polyclonal antibody production a mice 6 weeks, rats 6 weeks, rabbits 3 months, guinea pig 3 months , chicken 18-20 weeks, goats 6-7 months and sheep 7-9 months.
The third step is selection and preparation of the adjuvant. An adjuvant is a chemical that provokes a generalized activation of the immune system that can stimulates greater antibody production, is often mixed with the antigen prior to injection. ( Hendriksen and Hau 2003 ). Antigen-Adjuvant preparation have recommendations .such as to prepare mixtures aseptically, to carefully monitor the stability and quality of the emulsion, and to carefully select the injection route and volume, especially when oil adjuvants are used.
The fourth step is injection protocol. The choice of injection route is usually depended on the selection of animals and adjuvant, and also depends on character, quantity, and volume of the antigen. The common routes of injection for PAb production are subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.), and intravenous (i.v.). (Hendriksen and Hau (2003). Although it has advantages and disadvantages of different injection routes. The choice of animal species can also eliminate some of the injection routes. The i.d. route is not recommended in small rodents (mouse, rat, and hamster), and the i.p. route is not recommended in larger animals (rabbit and larger) ( Hendriksen and Hau 2003 ). After that, booster injection required to
Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen.
Materials and method
The lung cancer cell line A549 will be purchased from the American Type Culture Collection (ATCC). A549 cells will grow in RPMI 1640 (Sigma-Aldrich, Malaysia) containing 25 HEPES supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, Malaysia), penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (125 ng/ml).The cell must be maintained in a humidified atmosphere, 95% air, 5% CO2 at 37°C.
Low-passage number mycoplasma-free cells will seed at the concentration of 105 cells/mL and allow to grow for 48 hours . Before reaching confluence, cells will be harvested using a scraper and washed twice with 1X PBS (Bio-Rad, Singapore).
Cells will then lysed in a buffer containing 1% Triton X-100 (Sigma-Aldrich, Malaysia), 20 mM MOPS, 1 mM DTT (Thermo Fisher Scientific ,Singapore), 5 mM EDTA, 2 mM EGTA supplemented with phosphatase (20 mM sodium fluoride, 20 mM sodium pyrophosphate, 60 mM beta-glycerophosphate and 1 mM sodium orthovanadate) and protease (Roche cOmplete mini cocktail, Roche Diagnostics and 5 μM pepstatin A) inhibitors for 15 min on ice. The lysate will be sonicated three times for 5 sec and centrifuged at 16,100 g for 15 min at 4°C. Supernatants were kept and protein concentrations will be determined using standard BCA assay (Thermo Fisher Scientific,Singapore) .Three independent replicates for each cell line will be processed.
Samples were precipitated (100 μg) in 20% trichloroacetic acid (TCA) by incubation for 30 min at 4°C. After centrifugation at 4°C, 14,000 g, for 15 min, the pellets will be washed twice in ice-cold acetone, dried at room temperature and resuspended in 1X Laemmli buffer (10% glycerol, 62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 0.5% β-mercaptoethanol and bromophenol blue). Samples will be resolved on a 4–20% SDS-PAGE gradient gel and stained with coomassie blue colloidal G250 to visualize the gel bands.
Isolation of CKAP4 protein from SDS-PAGE:
After running the gel for 45 minutes, we will be able to see the bands based on molecular weight in the gel. After that, compare the bands with the molecular weight (MW) standard of proteins in the range 15-250 kDa. CKAP4 protein molecular weight is 63 kDa . So based on the position , by using a clean scalpel to cut off the protein-containing gel pieces.
Purification of CKAP4 protein:
Electroelusion is one of the technique to purify the protein from Polyacrylamide Gel Pieces. In this technique, protein-containing gel pieces are placed in an electroelution chamber, where the proteins are eluted from the gel matrix into a buffer solution using an electrical field and captured against a dialysis membrane with an appropriate molecular weight cut off.
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