Western Blot Test: Introduction, Principle, Procedure and Result Interpretation

Western blot test positive

Introduction of Western Blot Test 

Western blot test is a confirmatory test of  HIV-AIDS and it is positive as shown above image. It is also called immunoblotting and it is a technique for detecting specific proteins separated by electrophoresis by the use of labeled antibodies.  Blot is a technique for transferring DNA, RNA, and proteins onto a carrier so they can be separated, and often follows the use of gel electrophoresis. It is of the following types –

Southern blot: It uses for transferring DNA.

Northern blot: It uses for transferring RNA.

Western blot:  It is applicable for transferring protein and

Eastern blot: It is a biochemical technique used to analyze protein post-translational modifications (PTM) and is most often used to detect carbohydrate epitope.

Principle of Western Blotting/ Blot

It is an Immunoblotting technique that relies on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules. In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein. The sodium dodecyl sulfate (SDS) Polyacrylamide gel electrophoresis (PAGE) technique is a prerequisite for Western blotting. A technique for detecting specific proteins separated by electrophoresis by the use of labeled antibodies.

Test Requirements 

Provided in Test Kit-

  • Nitrocellulose Strips
  • Incorporated with HIV-1 viral lysate, a
  • specific HIV-2 envelope peptide and
  • specimen addition control band
  • Non-reactive control
  • Strong reactive control
  • Weak reactive control
  • Stock Buffer concentrate
  • Wash buffer concentrate
  • Conjugate
  • Substrate
  • Blotting powder
  • Incubation tray
  • Instruction for use
  • Forceps

Extra we need those are not provided-

  • Deionized or distilled water
  • Disposable gloves
  • Rocking platform (designed with a rocking speed range of 12 to 16
    cycles per minute, and which moves through a 5° to 10° tilt to wash
    membranes evenly)
  • Precision Pipettes ranging from 20µl  to 1000µl  dispensing volume
    and pipette tips of appropriate volume
  • Aspirator with sodium hypochlorite trap
  • 56°C water bath (optional)
  • Sodium hypochlorite for decontamination
  • Paper towel, adhesive tape, worksheet (non-absorbent white paper)

Test Procedure

It completes in following steps-

  1. Sample preparation ( protein extraction)
  2. Protein assay
  3.  Gel electrophoresis of protein: It may be performed by gel stain or
    by  protein transfer to a membrane
  4. Protein transfer to a membrane: membrane stain and blocking and washing
  5. Incubate primary antibody
  6.  Incubate secondary antibody
  7. It can be further completed by either chromogenic detection or
    chemiluminescent detection

Test procedure according to the manual of MP Diagnostics for HIV BLOT 2.2
WESTERN BLOT ASSAY. It is divided into two parts-

a.  Rapid Assay ( short procedure) and

b. Overnight Assay ( long procedure)

                   Rapid Assay Procedure

  1. Add 2 ml of diluted wash buffer to each well.
  2. Using forceps, carefully remove a required number of strips from the tube and place numbered sides up into each well. Include strips for Strong Reactive, Weak Reactive, and Non-Reactive controls.
  3. Incubate the strips for 1 to 2 minutes at room temperature (25 ± 3°C) on a rocking platform (speed of 12 to 16 cycles per minute). Remove buffer by aspiration. (Note: Do not allow the strips to dry. Failure may result in watery marks on developed strips for some specimens.)
  4. Add 2 ml of blotting buffer to each well. Tilt the tray slightly and add the specimen(s) where the buffer is collected at the lower end of each well as per the next step. Ensure that strip no. is at the higher end.
  5. Add 20 µl each of specimens to their respective wells, and followed by 20 µl each of Strong Reactive, Weak Reactive, and Non-Reactive Controls to their respective wells. The sequence of adding specimens or controls first is flexible.
  6. Cover the tray with the cover provided and incubate for 1 hour at room temperature on the rocking platform.
  7. Carefully uncover the tray to avoid splashing or mixing of samples. Tilt the tray to aspirate the mixture from the wells. Change aspirator tips between samples to avoid cross-contamination.
  8. Add 2 ml of diluted wash buffer. Wash each strip 3 times with 5 minutes soak on the rocking platform between each wash. (Note: Each wash cycle consists of dispensing 2 ml of diluted wash buffer, soaking time of 5 minutes, and aspiration.)
  9. Add 2 ml of the working conjugate solution to each well.
  10. Cover the tray and incubate for 1 hour at room temperature on the rocking platform.
  11. Aspirate conjugate from the wells. Wash as in step 8.
  12. Add 2 ml of substrate solution to each well.
  13.  Cover the tray and incubate for 15 minutes on the rocking platform.
    (Note: The reaction can be stopped before 15 minutes if all the bands are visible for high anti-HIV titer samples to avoid over development of bands and
    difficulty in reading.)
  14. Aspirate the substrate  and rinse the strips at least three times with reagent grade water to stop the reaction (A dark background can result if washing is
    insufficient at this step).
  15.  Allow strips to dry in the wells of the tray.
  16.  Mount strips on the worksheet (non-absorbent white paper). Do not apply adhesive tape over the developed bands. Observe the bands (See Interpretation of Results) and grade the results. For storage, keep the
    strips in the dark

OverNight Procedure of Western Blot

  1.  Add 2 ml of diluted wash buffer to each well.
  2.  Using forceps, carefully remove the required number of STRIPS from the tube and place numbered side up into each well. Include strips for Strong Reactive, Weak Reactive, and Non-Reactive controls.
  3. Incubate the strips for 1 to 2 minutes at room temperature on a rocking platform (speed of 12 to 16 cycles per minute). Remove buffer by aspiration. (Note: Do not allow the strips to dry. Failure may result in watery marks on developed strips for some specimens.)
  4. Add 2 ml of blotting buffer to each well. Tilt the tray slightly and add the specimen(s) where the buffer is collected at the lower end of each well as per the next step. Ensure that strip no. is at the higher end.
  5. Add 20 µl each of specimens to their respective wells, and followed by 20 ul each of Strong Reactive, Weak Reactive, and Non-Reactive Controls to their respective wells. The sequence of adding specimens or controls first is flexible.
  6. Cover the tray with the cover provided and incubate overnight (16 – 20 hours) at room temperature on the rocking platform.
  7. Carefully uncover the tray to avoid splashing or mixing of samples. Tilt the tray to aspirate the mixture from the wells. Change aspirator tips between samples to avoid cross-contamination.
  8. Add 2 ml of diluted wash buffer. Wash each strip 3 times with 5 minutes soak on the rocking platform between each wash. (Note: Each wash cycle consists of dispensing 2 ml of diluted wash buffer, soaking time of 5 minutes, and aspiration.)
  9. Add 2 ml of the working conjugate solution to each well.
  10. Cover the tray and incubate for 30 minutes at room temperature on the rocking platform.
  11. Aspirate conjugate from the wells. Wash as in step 8.
  12. Add 2 ml of substrate solution to each well.
  13. Cover the tray and incubate for 15 minutes on the rocking platform. (Note: The reaction can be stopped before 15 minutes if all the bands are visible for high anti-HIV titer samples to avoid over development of bands and difficulty in reading.)
  14. Aspirate the substrate and rinse the strips at least three times with reagent grade water to stop the reaction (A dark background can result if washing is
    insufficient at this step).
  15. Allow strips to dry in the wells of the tray.
  16.  Mount strips on the worksheet (non-absorbent white paper). Do not apply adhesive tape over the developed bands. Observe the bands and grade the results. For storage, keep the strips in the dark.

Result Interpretation of Western Blot

 

Pattern                                                                        Interpretation

  1. No viral-specific bands present                            Negative
  2. Detection of p17 antibodies
    ONLY, no other bands                                                Negative
  3. Detection of 2 ENV
    (gp160/gp41and gp120) and 1
    GAG (p17, p24, p55) or 1 POL                                HIV-1 Positive
    (p31, p51, p66)
  4. Detection of 2 ENV
    (gp160/gp41 and gp120) and                        HIV-1 Positive with HIV-2 Indicated
    1 GAG (p17, p24, p55) or 1
    POL (p31, p51, p66) and HIV-2
    the specific band is visible
  5. Any viral-specific bands present
    but the pattern does not meet                                      Intermediate 2
    criteria for POSITIVE
  6.  Any viral-specific bands present
    but the pattern does not meet                                      Intermediate 2 with
    criteria for POSITIVE but HIV-2                              HIV-2 Indicated
    the specific band is visible.

Applications of Western Blot

  • Western blot analysis can detect target protein which is as low as 1 ng due to the high resolution of the gel electrophoresis and strong specificity and high sensitivity of the immunoassay. It is applicable in the fields of molecular biology, biochemistry, immunogenetics, and other molecular biology disciplines.
  • It uses for the identification of a specific protein in a complex mixture of proteins.
  • Western blot is most widely used as a confirmatory test for the diagnosis of HIV.
  • It is also used as the definitive test for Bovine spongiform encephalopathy (BSE), commonly referred to as mad cow disease and its causative agent is prion which is the only protein in nature that may be either living or non-living.
  • Western blotting also uses for some forms of Lyme disease testing.
  • Demonstration of specific antibodies in the serum for diagnosis of neurocysticercosis and tubercular meningitis.

Advantages of Western Blot

Advantages of Western blot/blotting are as follow-

  • Western blot is a more specific test for detection of HIV than ELISA which is a non-specific test.
  • It is also applicable for the detection of a single protein from a mixture of proteins while giving information about the size of the protein and so is more specific.
  • It also gives information about how much protein has accumulated in cells.
  • Western blot test is referred to as the Gold Standard.

Disadvantages of  Western Blot

  • Quicker degradable proteins are not detected properly.
  • The test is the time is taken and costly as compared to rapid tests of HIV/AIDS.

Further Reading

  1. https://www.who.int/diagnostics_laboratory/evaluations/160404_final_public_report_0198_071_00_v2.pdf?ua=1
  2. https://en.wikipedia.org/wiki/Eastern_blot
  3. https://advansta.com/products/Advanstas-Step-by-Step-Guide-to-Western-Blots
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
  5. https://www.enzolifesciences.com/sciencecenter/technotes/2019/december/what-are-the-differences-between-northern-southern-and-westernblotting?/
  6. https://www.sigmaaldrich.com/technicaldocuments/protocols/biology/western-blotting.html
  7. https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-principle
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