Section: 802, (Thursday)

Group: 4

Names: John Gibson, Kyle LaPlant

Instructor: S. Royan, PhD

Date: Oct 10, 2024

University Of Massachusetts,

Lowell, Fall 2024

MicroElisa Assay Of Mouse Immunoglobin Production Against Soluble Antigen

Contributions: This Entire Report: John Gibson

 

Objective

This experiment aimed to use enzyme-linked immunosorbent assay with microtiter strips (MicroELISA) to assay a mouse’s immunoglobulin production against soluble protein antigens after two immunizations. The assay results were compared to the textbook understanding of the specific immune response after multiple exposures to the same antigen (1).

Material And Method

Mouse Immunization

A CD-1 breeder mouse was given a peritoneal injection with commercially sourced rabbit immunoglobin IgG in Complete Freund’s Adjuvant (CFA) to elicit a first-exposure immune response (2). The CFA encompassed mineral oil and dry-inactivated mycobacteria particles. A pre-exposure bleed serum (PS) was obtained immediately before the first injection. 

After one week, the immunization bleed serum (IS) was collected, and a second exposure injection was performed on the same mouse with Incomplete Freund’s Adjuvant (IFA), which was CFA without the mycobacteria particles. 

After one more week, a third blood collection was performed for the re-immunization bleed serum (ISR). All serum was stored in a refrigerator before the ELISA assay.

Microtiter  Strip Preparation

Two 8-well flat-bottom microtiter assay strips were coated with the same rabbit immunoglobin IgG, 10 µg/mL in phosphate buffer saline at pH 7.4 (PBS), one week before the MicroELISA assay. The coating volume was 100 μL. The coating was incubated at room temperature for 2 hours. After emptying the coating solutions, the 16 wells were all stored with a sterile 1% Bovine Serum Albumin (BSA) blocker in PBS (pH 7.4) solution to block non-specific bindings. Strip 1 was stored with 100 μL PBS+BSA solution, while strip 2 was stored with 200 μL PBS+BSA solution at 4°C. 

MicroELISA Immuno Binding Well Loading

Positive control (PC) antibodies for the same rabbit IgG were commercially sourced. Anti-mouse goat antibodies conjugated with horse radish peroxidase (HRP) were commercially obtained. PBS (pH 7.4) with Tween20 (PBST) solution was prepared by giving 3 drops of Tween20 in 50 mL PBS. 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) 0.25 mg/ml was commercially obtained. 

 The PBS+BSA blocking solutions in the 16 wells of the 2 strips were removed, and the 16 wells were each washed with 200 μL PBST 3 times and then emptied.

The first microtiter strip was loaded with contents in Table 1 below for primary antibody binding of the immunized bleed serum (IS) and control groups; the second microtiter strip was loaded with contents in Table 2 below for primary antibody binding of the re-immunized bleed serum (ISR). The microtiter strips were incubated at room temperature for 30 minutes. 

Table 1. MicroELISA Strip Loading For First Immunization Response

Reference: Page 38, Figure 12 (2)

Legend: Antigen is rabbit immunoglobulin IgG. Sera are pre-exposure serum (PS), 1-week after immunization (IS), and commercially sourced positive control (PC) of mouse antibodies against the same rabbit immunoglobulin IgG. PBS-Tween is a biological phosphate buffer with 3 drops of Tween20 in a 50 mL solution. The plus sign “+” denotes the inclusion of the reagent component in the microtiter strip’s well.

Table 2. MicroELISA Strip Loading For Second Re-Immunization Response

Reference: Page 38, Figure 12 (2)

Legend: ISR serum was post-exposure serum 2 weeks after the first explore and 1 week after the second re-immunization (ISR). Antigen and PC same as Table 1.

After the primary antigen binding incubation, the wells’ contents were removed, wells were washed 3 times as before, and secondary antibodies (anti-mouse goat antibodies conjugated with HRP) were loaded into all 16 wells, 100 μL each. The microtiter strips were incubated at room temperature for 1 hour. 

MicroELISA Chromogenic Reaction Setup

Before ABTS chromogenic reactions were performed to elucidate secondary antibody binding status, all wells were washed with PBST 2 times as before, and then PBS-only washing 2 times, each with 200 μL of PBS for each well.

Before dumping the final washing PBS-only fluid from the strips, two volumes of 1.8 mL ABTS were activated by adding 100 μL H2O2 to each volume. Then, the wells were immediately emptied of PBS-only wash solution and loaded with activated ABTS, 100 μL. The wells were photographed, and the absorbance of 412 nm wavelength light (A412) was measured within 5 minutes.

Data (3)

Figure 1 below shows a photograph of the first MicroELISA strip (with bleed sera 1 week after the single immunization) after about 5 minutes of ABTS chromogenic reaction with HRP and H2O2. 

The titer of IS immunization bleed serum was 100, as indicated by the blue hue in wells 5 and 6.

Figure 2 below shows a photograph of the first MicroELISA strip (with bleed sera 2 weeks after the first immunization and 1 week after the second immunization) after about 5 minutes of ABTS chromogenic reaction with HRP and H2O2. 

The titer of IS immunization bleed serum was 100, as indicated by the blue hue in well 7. The blue hue in well 8 was indistinguishable from the hue in well 2, a negative blank.

The measured light absorbance at the wavelength of 412 nm (A412) is shown in Table 3. 

Table 3. A412 Absorbance Of ELISA Assay With ABTS Reaction In Strip 1

Reference: (3)

Legend: Blank refers to the measured absorbance of wells with only PBS and no serum. Average is the mean absorbance value for repeated reactions with the same serum, reagents.

Average - Blank was the absorbance value for comparison between different wells reactions. 

The measured light absorbance at the wavelength of 412 nm (A412) is shown in Table 4 below.

Table 4. A412 Absorbance Of ELISA Assay With ABTS Reaction In Strip 2

Reference: (3)

Legend: Blank, Average, and Average - Blank same as Table 3.

Result

As expected, the chromogenic reaction produced a blue hue, visible in immunized sera wells 4, 5, and 6, and positive control wells 7 and 8. The 1:100 dilution of wells 5 and 6 is the highest among the immunized sera in strip 1 (Figure 1), indicating the titer of the single-immunization bleed serum at 100. 

The corresponding A412 measurements of strip 1 show absorbance between -0.02 of the pre-immunization bleed serum and 0.366 of the single immunition bleed serum (Table 3). The 0.348 absorbance (after subtracting the bland) of the 1:100 dilute immunized sera also indicated the titer of the single-immunization bleed serum at 100.

As expected, the chromogenic reaction produced a blue hue, visible in re-immunized sera wells 4 through 7 and positive control well 3 (Table 4). The 1:5000 dilution of well 7 is the highest among the re-immunized sera in strip 2 (Figure 2), indicating the titer of the single-immunization bleed serum at 5,000. 

The corresponding A412 measurements of strip 2 show absorbance between 0.065 of the re-immunization bleed serum at 1:10,000 dilution and 0.672 at 1:10 dilution (Table 4). The significant absorbance of 0.109 of re-immunization bleed serum at 1:5,000 dilution implicated the titer of the re-immunization bleed serum at 5,000. 

The blanks and the negative control absorb 412 nm light between 0.143 and 0.188 in wells 1 and 2 of both strips and well 3 of strip 1 (Table 3 and 4), with a 0.045 difference between the highest and the lowest. 

The absorbance of 0.065 of re-immunization bleed serum at 1:10,000 dilution is close to the fluctuation between blanks and negative control.

Discussion

ELISA assay detects an antigen or an antibody by specific immuno-binding of immunoglobulin to antigen and then detects the binding with an antibody-conjugated enzyme’s reactions. Two common forms of ELISA assay were direct antigen detection and indirect antigen detection. In direct antigen detection, the enzyme was conjugated to the primary antibody of the antigen in question; in indirect antigen detection, the enzyme was conjugated to a secondary antibody that binds to the primary antibody. The primary antibody binds to the antigen in question. The enzyme facilitates different reactions in different experiment setups, such as luminol oxidation that makes the substrate emit light or chromogenic reactions that change the substrate’s color. 

In this experiment, the indirect ELISA assay was utilized, where the primary antibodies were produced by immunization of a mouse with rabbit IgG antigen, and the secondary antibodies were commercially available anti-mouse goat antibodies that bind to mouse immunoglobin at the variable region of the heavy chain-light chain dimmer of the goat antibodies. The secondary antibodies were conjugated with horseradish peroxidase (HRP) for a chromogenic reaction, changing the color of the ABTS substrate to blue. The color change manifests as absorbing light at 412 nm wavelength.

Comparing the two strips’ negative control wells, the absorbance of light at a wavelength of 412 nm was very similar, with only about 11% difference in average between 0.1630 and 0.1835 in wells 1 and 2 of strip 1 and strip 2 respectively (Tables 3 and 4). This indicated that contamination, if any, levels between the two strips were very similar. Furthermore, wells 1 and 2 of both strips were colorless (Figures 1 and 2), meaning that the concentration of antibodies in them was minimal due to the lack of goat anti-mouse antibodies with conjugated HRP. This means that contamination was negligible in this experiment.

Comparing the two strips’ positive control wells, the A412 was very similar after subtraction of the absorbance with the blank (PBS-Tween only), with only about a 32% difference in average absorbance between 0.358 and 0.524 in wells 7 and 8 of strip 1 and strip 2’s well 3 respectively (Tables 3 and 4). This indicated that the chromogenic reaction intensity was similar between the 2 strips. Furthermore, the color of wells 5 and 6 of strip 1 was similar to that of wells 7 and 8 of strip 1’s positive control (Figures 1 and 2), meaning that the chromogenic ABTS reaction with HRP and H2O2 was correctly set up in both strips.

Comparing PS preimmune and IS immunized sera, at 1:10 dilution, IS’s A412 at 0.366 (after subtraction of blank) was a positive absorbance, the opposite sign of the PS’s A412 at -0.02 (after subtraction of blank) that was negligible. This indicated that the first immunization in the mouse resulted in antibody production against the rabbit immunoglobulin IgG. 

Comparing PS preimmune and ISR re-immunized sera, at 1:10 dilution, ISR’s A412 at 0.672 (after subtraction of blank) was a positive absorbance, the opposite sign of the PS’s A412 at -0.02 (after subtraction of blank) that was negligible. This indicated that the two immunizations in the mouse resulted in antibody production against the rabbit immunoglobulin IgG.

Comparing IS immunized and ISR re-immunized sera at 1:10 dilution, ISR’s A412 at 0.672 (after subtraction of blank) was 183% of IS’s A412 at 0.366 (after subtraction of blank). This indicated that the second immunization in the mouse resulted in additional antibody production against the rabbit immunoglobulin IgG. This matches the textbook understanding of the specific immune system’s production of antibodies with multiple exposures to the same antigen (1). 

Comparing IS immunized and ISR re-immunized sera at 1:100 dilution, ISR’s A412 at 0.385 (after subtraction of blank) was also higher than IS’s A412 at 0.348 (after subtraction of blank). This also implicated that a second immunization in the mouse produced additional antibodies against the rabbit immunoglobulin IgG.

The titer of the first immunized bleed serum was 100, indicated qualitatively by the blue hue in wells 5 and 6 of strip 1 (Figure 1), which had a dilution factor 1:100 from the serum. This titer level quantitatively corresponded to the average absorbance of 0.348 after subtracting the blank absorbance (Table 3). 

The titer of the re-immunized bleed serum was 5000, indicated qualitatively by the blue hue in well 7 of strip 2 (Figure 2), which had a dilution factor of 1:5000 from serum. This titer level quantitatively corresponded to the absorbance of 0.109 after subtracting the blank absorbance (Table 4). 

The statistical significance of 0.065 absorbance of re-immunization bleed serum at 1:10,000 dilution was unclear. The blanks and negative control had absorbance differences as large as 0.045, implicating the confident titer level of the re-immunization bleed serum at 5,000.

The microtiter strips storage PBS+BSA blocking solution volume difference, 100 μL in strip 1 and 200 μL in strip 2, didn’t significantly affect the antibody bindings to rabbit immunoglobin. In particular, for the blank wells, the goat anti-mouse IgG conjugated with HRP in strip 1 did not elicit a stronger binding than in strip 2 (strip 1’s wells 1 and 2’s A412 were not higher than the counterparts’ in strip 2 in Tables 3 and 4), meaning that the 200 μL BSA blocker volume covering the inner wall of the wells above the 100 μL level did not offer any more blocking between antibodies and the plastic material of the microtiter strips.

Conclusion

The experiment successfully demonstrated the immune response of the mouse, as expected. In particular, the second booster shot increases antibody production, as expected in textbook understanding (1). The mouse immunization injections were effective. Contamination was negligible, and the chromogenic reaction setup was effective.

References 

1. Juris, Stephen. Immunology. New York, Oxford University Press, 2022.

2. Royan, S.V. & A. Alton. (2024). Immunology Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.

3. Gibson, John. (2024). Immunology Laboratory Notebook Fall 2024. [Unpublished Immunology Laboratory Notebook]. Department of Biological Sciences, University of Massachusetts, Lowell.