Question

Please help with all questions with regards to ELISA

1. Explain how one would go about preparing mouse anti-Tomato Spotted Wilt antiserum (5).

2. Explain how one would go about preparing a goat anti-mouse-Fc = alkaline phosphatase conjugate (9).

3. Please explain how to prepare a 1:20 dilution, showing an example (3)

4. Why is it necessary to wash the wells between each step (2)

5. Why was a goat anti-rabbit Fc specific conjugate used in the ELISA, rather than a goat anti-rabbit IgG specific conjugate? What would have happened if the goat anti-rabbit IgG conjugate was accidently used (2)?

6. Explain the need for the specific controls included in the test (5).

Answer 1

1. Depending on the test kits, 1:1000 or 1:200 dilutions are prepared of the mouse anti-Tomato Spotted Wilt antiserum.

2. Alkaline phosphatase-conjugated goat anti-mouse Fc specific antibody was used as a secondary antibody in ELISA assays at a dilution of 1:1000 in PBS (Phosphate Buffer Saline)/0.1% Tween and 1% BSA (Bovine Serum Albumin) for 1.5 hours at 37°C.

3. Preparation of 1:20 dilution : Here the solute will be 1 and the solvent will be 20 part of the whole solution.

Suppose the final volume of solution (Chemical + Water) will be 100 ml.

to calculate the volume/amount of solute (chemical): first the ratio will be obtained by taking the solute (chemical) part as numerator and solvent part as denominator, here it will be 1/20. Then the volume of solute will be calculated by multiplying the final volume to the ratio of solute:solvent. Here, 100 x 1/20 = 5 ml chemical.

So, the volume of solvent will be= 100-5 =95 ml water.

4. During ELISA , wells are properly washed with as solution between each steps in order to remove any unwanted, non specific background such as unbound proteins or antibodies, so that these unbound particles will not hinder the final ELISA reading and indicate the actual quantity of antigen in the sample.

5. The use of goat anti-rabbit Fc specific conjugate in ELISA expresses strict isotypic (class) specificity when react with antiserum. This specificity is because of the the Fc subunit of IgG.  Here, IgG is an immunoglobulin monomer consisting of two (gamma) heavy chains and two light chains. Each IgG molecule contains two antigen binding domains and a single effector (Fc) domain.

Inter-species cross-reactivity is a normal feature of antibodies to immunoglobulins, since Ig of different species frequently share antigenic determinants. So, the use of the goat anti-rabbit IgG conjugate can lead to cross reactivity, resulting into non specific result.

6. The most basic control of ELISA is the accurate blank solution so that the comparison with the blank will lead to correct analysis. Additional controls are needed to provide a comparison to real world physiological conditions and a control mechanism for assurance that the assay continues to provide accurate results.