• Immunoblotting (Western Analysis)

Immunoblotting (is also called «Western blotting») is a method for identifying a particular antigen in a complex mixture of proteins. The complex mixture of proteins is subject to sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE). This separates the proteins according to molecular size. The gel is then covered with a membrane (usually a sheet of nitrocellulose), and the proteins are «transferred» by electrophoresis to the membrane. The nitrocellulose membrane (blot) acquires a replica of the proteins separated by SDS-PAGE. During the transfer, the SDS is largely removed from the proteins and, at least for some proteins, there is refolding into a conformation such that antibodies can react with the proteins on the membrane.

The nitrocellulose is then incubated with either a radioactive or enzyme-labeled antibody in a direct test or in an indirect test with antibody followed by a labeled antiglobulin. The protein antigen then becomes visible as a band on the membrane. None of the other proteins in the mixture are detected.

Practical work

1. The Mechanism of ELISA for Detecting Hbs Ag in the Patient’s Serum. Specific antibodies against Hbs Ag are fixed to a solid surface (commercially prepared plastic microtiter plate), and then the patient’s serum is added.

NOTE: Patient’s serum is usually examined to detect the antibodies. But hepatitis B is also known as parenteral hepatitis. Therefore viral antigen Hbs Ag can be detected in the serum and blood.

Therefore, the direct ELISA («sandwich» modification) is performed.

If Hbs Ag is present in the tested serum, it will bind to antibody adsorbed to a well and be fixed to the container. The serum and all unbound components are washed away before the next step. Enzyme conjugate (enzyme-linked specific antibodies against Hbs Ag) is then added. The conjugate is prepared by chemically linking the indicator enzyme (in this case, peroxidase) with specific anti-Hbs antibodies. This conjugate will bind to the fixed antigen-antibody complex. If both the antibody adsorbed to the well and the antibody known to be specific for Hbs Ag have reacted with the antigen, a «sandwich» will have formed, with the antigen between two antibody molecules. This in turn fixes peroxidase to the microtiter plate. Unbound enzyme conjugate is washed away before the next step. The substrate for enzyme action, (hydrogen peroxide), and a colorless chemical indicator (chromogen) are then added. Peroxidase converts H₂O₂ to H₂O and O₂ . In the presence of O₂ , the indicator changes color, indicating a positive reaction. Serum of the health person does not contain Hbs Ag and consequently fail to provide bridges that link the enzyme conjugate to the fixed antibodies. All peroxidase is therefore removed during the second wash, and no O₂ is generated. Negative reaction therefore shows no color (see Fig. 10).

The Procedure of Detecting Hbs Ag in the Patient’s Serum by ELISA. The ELISA is performed according to Table 14-1.

Table 14-1

The Scheme of the ELISA (solid phase modification)

Ingredients	Test	«Positive control»						«Negative control»
	A1 A2	B1 B2	C1 C2	D1 D2	E1 E2	F1 F2	G1 G2	H1 H2
1. Buffer A (ml)	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Incubation at room temperature for 10 min.
2. The patient’s serum (ml)	0.1	--	--	--	--	--	--	--
3. Standard antigen 100 μg/ml (ml)	--	0.1	0.1	0.1	0.1	0.1	0.1 pour out	--
4. Reagent «negative control» (ml)	--	--	--	--	--	--	--	0.1
Incubation at 37oC for 20 min.
5. Conjugate (ml)	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Incubation at 37oC for 20 min.
6. Substrate (ml)	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Incubation at room temperature for 20 min.
7. 50% sulfuric acid (ml)	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Results: (color/no color)
Conclusion: the ELISA is ________________(positive/negative). The result means, that Hbs Ag is (not) detected in the patient’s serum. The concentration of HBs Ag in the patient’s serum is _______ μg/ml.

Step 1.

1. Take the special plastic microtiter plate with numerous shallow wells. The anti-Hbs antibodies are adsorbed to its wells. Choose two vertical rows for performing the test.

2. Inoculate 0.1 ml of buffer A into each well in each vertical row (from A₁ to H₁ and from A₂ to H₂). Allow the plate to stand for 10 min. at room temperature.

3. Add equal amount (0.1 ml) of patient’s serum to the wells #A₁ and #A₂.

4. Add at 0.1 ml of standard antigen (initial concentration 100 μg/ml) to the wells B₁ and B₂, mix thoroughly and transfer 0.1 ml of the mixture into the next wells (C₁ and C₂ accordingly). Prepare the serial two-fold dilution in the next wells (from D₁ to G₁ and from D₂ to G₂). Pour out 0.1 ml of the mixture into the conteiner with disinfectant from the G₁ and G₂ wells.

5. Add at 0.1 ml of «negative control» to the wells H₁ and H₂.

6. Place the microtiter plate in the 37^oC incubator. Allow plate to stand for 20 min.

Step 2.

1. Rinse off the plate with tap water.

2. Pipette 0.1 ml of conjugate (enzyme-labeled antiserum) into each well in each vertical row (A ₁- H₁ and A₂ - H₂).

3. Incubate the plate at 37^oC for 20 min.

Step 3.

1. Wash off the plate with tap water three times before the next step.

2. Add 0.1 ml of substrate solution into each well (see Table 14-1).

3. Allow the plate to stand at room temperature for 20 min.

4. Add 0.05 ml of 50 per cent sulfuric acid into each well to stop the reaction.

BE CAREFUL: The step D is performed by lab technician!

5. Read the results visually. To do this, examine the color in the tested wells (A₁ and A₂), compare the color in these wells with the color of H₁ and H₂ (negative control). Compare the intense of color in the A ₁ and A₂ (if the reaction in the tested well produces a visible color change) with the color in the wells with diluted standard antigen ( B₁ - G₁ and B₂ - G₂).