Principle:

• Western blotting technique is used for identification of particular protein from the mixture of protein.
• In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.

Procedure/Steps:

1. Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.

Step I: Extraction of Protein

• Cell lysate is most common sample for western blotting.
• Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as tissue preparation.
• To prevent denaturing of protein protease inhibitor is used.
• The concentration of protein is determined by spectroscopy.
• When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which helps to sink the sample in well.
• Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.

Step II: Gel electrophoresis

• The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis.
• The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of these all.
• The small size protein moves faster than large size protein.
• Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.

Step III: Blotting

• The nitrocellulose membrane is placed on the gel. The separated protein from gel get transferred to nitrocellulose paper by capillary action. This type of blotting is time consuming and may take 1-2 days
• For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can be used.
• In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.

Step IV: Blocking

• Blocking is very important step in western blotting.
• Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary antibody the membrane is non-specifically saturated or masked by using casein or Bovine serum albumin (BSA).

Step V: Treatment with Primary Antibody

• The primary antibody (1° Ab) is specific to desired protein so it form Ag-Ab complex

Step VI: Treatment with secondary antibody

• The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody.
• Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex.

Step VII: Treatment with suitable substrate

• To visualize the enzyme action, the reaction mixture is incubated with specific substrate.
• The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the membrane.
• Western blotting is also a quantitative test to determine the amount of protein in sample.

Application:

1. To determine the size and amount of protein in given sample.
2. Disease diagnosis: detects antibody against virus or bacteria in serum.
3. Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
4. Useful to detect defective proteins. For eg Prions disease.
5. Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and Herpes

Western blotting technique: principle, procedure and application

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HIV Replication

• HIV uses CD4 molecule as a receptor which is present in CD4+ cells such as T-lymphocytes, macrophages, monocytes, dendritic cells other and Antigen presenting cells.

• A second co-receptor in addition to CD4 molecule is required for HIV to gain entry into host cell. CCR5 for macrophage tropic HIV and CXCR4 for T-lymphocyte tropic HIV.

• HIV first binds to CD4 molecule mediated by surface glycoprotein gp120 and then to the corresponding co-receptors CCR5 or CXCR4. This binding brings conformational change in viral envelope inducing the binding of gp41 of virus into host cell membrane and triggering the entry of virus into host cell.

• Once the fusion of virus takes place with the host cell, viral RNA is released into the cytoplasm.

• Viral RNA is then used to synthesize ds DNA by the enzyme reverse transcriptase (RNA dependent DNA polymerase). The ds DNA is then circularized and enter into host cell nucleus.

• This circular ds DNA get integrated with host genome and the process is catalyzed by the enzyme integrase. This type of integrated viral DNA into host DNA is known as Pro-virus.

• Once the viral DNA is integrated, infection of HIV is permanent.

• The HIV virus may then enter into latency or enter into productive cycle.

• In productive cycle, the pro-virus DNA is then transcribed into mRNA by host RNA polymerase and finally translated to viral proteins. These viral proteins are processed to form virion components which are then assembled.

• The progeny virus now mature and release by budding.

• During budding, it acquire envelope.

• The released progeny virus infects suitable host cell and continues the productive cycle.

Pathogenesis of HIV/AIDS

Pathogenesis of HIV can be described under following headings.

1. Mode of transmission:

HIV infection is acquired through one of the following routes.

• Sexual contact; infected partner
• Parental route; infected needles or syringes
• Congenital route; infected mother giving birth

2. Primary infection: (acute infection):

• Once HIV enters the body through any of those routes they bind to CD4+ cells with their glycoprotein spikes and infect the cell. Thus the infection is established.
• HIV multiply in mucosa and there is 4-11 days between mucosal infection and initial viremia. Viremia is detectable for about 8-12 weeks.

3. Dissemination of virion to lymphoid organs:

• Virus from blood is then disseminated throughout the body and seeded in lymphoid organs.
• In this stage, it leads to acute mononucleosis like syndrome
• The CD4 T- cells count drop significantly during this time.

4. Clinical latency:

• An immune response to HIV develops 1 week to 3 months after infection, which leads to significant drop in viral load and rebounce of CD4 T-cells.
• The immune response however is unable to clear the HIV completely.
• The HIV infected cells persists in lymph nodes leading to clinical latency.

5. Elevated HIV level:

• The period of clinical latency lasts as long as for 10 years.
• During latency virus replication continues. Consequently patients develop constitutional symptoms and clinical diseases such as opportunistic infections, neurological disorder and neoplasm.

6. Clinical disease and death:

• The duration between primary infection and progression to clinical disease is 10-12 years while death occurs usually within 2 years after unset of clinical disease ie. AIDS in untreated cases.

Clinical manifestation of HIV/AIDS

1. Acute retroviral syndrome (ARS):

• It can occurs few days to few weeks after primary infection.
• It is found in 50-70% of infected patients.
• It is characterized by clinical signs of immune activation and multi system dysfunction

Symptoms includes;

• acute mononucleosis like syndrome
• fever
• generalized lymphadenopathy
• sore throat
• fatigue
• weight loss
• rashes,
• nausea
• night sweat
• occasionally diarrhea
• pancreatitis
• bacterial sepsis
• epiglottitis
• self limiting neurological disorder,
• meningitis,
• encephalitis.

All these symptoms resolves within 5-30 days.

2. Clinical latency and incubation to AIDS

• ARS is followed by long period of clinical latency which is free of any clinical symptoms
• Clinical latency is maintained because of host immune response against HIV
• The median time to develop AIDS in untreated cases is 10-12 years.
• 5-10% of patients are rapid progressors and they develops AIDS within 2-3 years
• Similarly, 5-10 % other patients are non-progressors and they become free of symptoms after 7-8 years.
• In congenital infection clinical symptoms usually appears by 2 years of age and death occurs in next 2 years.

3. AIDS:

• Significant drops in CD4 T-cells count (below 200 cells/µl) leads to AIDS.
• AIDS results in loss of ability to respond to infections.
• Loss of immune response immune response develops opportunistic infection and tumors
• Other clinical symptoms are; Tuberculosis, Parasitic infection, Hepatitis, oral hairy leukoplankia, Kaposi sarcoma, Dementia complex and other neurological symptoms.

Lab diagnosis:

1. Serology: detection of anti-viral antibody using ELISA. This test detects serum antibody against gp120, gp41, P24 etc. The first positive result must be confirmed by at least 2 other different assays with different viral antigen.

2. Western blotting: It is a confirmatory test.

3. RT-PCR: detection of RNA and DNA sequence of HIV

4. Virus isolation: culturing on CD4 + T-lymphocyte cell line

HIV/AIDS: Replication, pathogenesis, clinical manifestation and lab diagnosis

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