ELISA test: How to detect rabbit anti-BSA antibodies?

The ELISA test (acronym for Enzyme Linked ImmunoSorbent Assay) is an immunological test intended to detect and / or measure a protein in a biological fluid. Its principle is indicated on the ELISA test page: detection of bovine beta-lactoglobulin.

Microtiter strips in their support

In the test presented here, an antibody directed against bovine serum albumin (BSA) is sought in a rabbit serum. The wells of a microplate were lined in advance with BSA which attaches to the plastic of the wells by electrostatic interactions.

BSA allows the specific capture of anti-BSA antibodies possibly present in the sera tested. The anti-BSA antibodies are then revealed by a tracer anti-rabbit immunoglobulin antibody conjugated to peroxidase. The reaction of the enzyme with tetramethylbenzidine gives a blue derivative whose concentration is proportional to that of the antibodies detected.

Protocol

The ELISA kit includes the well strips covered by the BSA and their support as well as all the necessary reagents, either ready to use (PBS-tween buffer), or to be reconstituted with distilled water or phosphate buffer salt ( Phosphate Buffer Saline, PBS) from concentrated solutions or freeze-dried products according to the instructions given in the instructions for use. If it is desired to make an assay of the antibody in an unknown serum, it is advisable to carry out beforehand a dilution range of the initial antibody solution so as to construct a calibration curve. The mother antibody solution, obtained by diluting the antibodies supplied in 15 ml of PBS, comprises 17 μg of rabbit anti-BSA antibody per ml . We will call it C1. 

In all cases, the procedure is similar:

  1. Place 80 µL of test serum or 80 µL of different antibody dilutions in the wells and incubate for 15 min at room temperature. 
  2. Empty the wells by inversion and rinse twice by filling the wells with the PBS-tween buffer and emptying them by inversion.
  3. Add 80 µL of the detection antibody solution to the wells.
  4. Incubate 15 min at room temperature.
  5. Empty the wells by inversion and rinse twice with PBS-tween buffer.
  6. Add 80 µL of the developer solution ( tetramethylbenzidine , TMB) and incubate 5 min at room temperature.

The range is carried out by serial dilutions to 1/2 as indicated in the table below:

Volume of antibody to be collected ( mL )Volume of PBS to be added ( mL )Last nameConcentration
(µg.L -1 )
7.5 of C17.5C28.50
7.5 of C27.5C34.25
7.5 of C37.5C42.12
7.5 of C47.5C51.06
7.5 of C57.5C60.53
7.5 of C67.5C70.26
7.5 of C77.5C80.13

Range of dilutions

Dilution range of rabbit anti-BSA antibody
(before and after addition of TMB)

The presence of anti-BSA antibodies results in the appearance of a blue coloration, the greater the higher the antibody concentration.

Results

The quantification of the results of ELISA tests is usually carried out with a microplate reader. If such equipment is not available, which is most often the case in high school laboratories, quantification remains possible.

Either with a colorimeter after having poured the content of the wells to be analyzed into spectrophotometer microcuvettes, or by taking advantage of the possibilities of data processing: in fact, the intensity of the coloration obtained in the different wells can be evaluated on a digital image of results with appropriate software.

If you simply want to observe the result of a qualitative test, you can use the antibody stock solution as a positive control as well as various negative controls (normal rabbit serum, well without tracer antibody, well without TMB …) .

Some results after treatment with HCl

By adding 80 μL of hydrochloric acid to 1 mol.L-1 in each well, the enzymatic reaction is stopped and the coloration turns yellow, which makes it possible, if necessary, to quantify the reaction by colorimetry at 450 nm.

To precisely determine the concentration of anti-BSA antibody in an unknown serum, it is necessary to construct a calibration curve from the results obtained with the dilution range of the mother antibody solution presented above.

Superimposition of the results of the standard range and the corresponding densitometric profile

For this, a digital image of the results in .bmp format is first obtained with a scanner or a camera and the densitometric profile is calculated with the Digispec or Mesurim processing software. The data are then translated graphically with any spreadsheet-graphing software. The results are presented in the diagram opposite.

Densitometric profile of a strip treated with HCl

The same type of treatment can also be carried out after treatment of the wells with HCl as shown in the assembly opposite.

The different levels of the profile are then quantified using the functions of the spreadsheet or graphical software. The standard curve, representing the color density of the wells as a function of the antibody concentration, is then plotted. For the range of dilutions used, the relationship between color density and antibody concentration is linear as shown in the figure below corresponding to the results obtained for the C1-C8 range without final treatment with HCl.

Therefore, the determination of the antibody concentration in other wells can be determined graphically: after having measured in the same way on a digital image the coloring density of the wells containing an unknown concentration of antibody, the measured density is plotted on the right. The concentration sought corresponds to the abscissa of these points.

Calibration curve
(the point labels correspond to the relative density of the color in the wells)