Section: 802, (Thursday)

Group: 4

Names: John Gibson

Instructor: S. Royan, PhD

Date: Oct 31, 2024

University Of Massachusetts,

Lowell, Fall 2024

Hemagglutination And Ouchternoly Assay Reactions Between Antigens And Antibodies

 

Objective

This experiment aimed to use the principle of hemagglutination to verify a mouse’s immunological response to non-soluble antigens on the surface of sheep red blood cells and to verify the Ouchternoly precipitation pattern between the full-set rabbit immunoglobulin G, M, A, D, and E antigens and 2 different antibody serum mixtures.  The assay results were compared with textbook and literature descriptions (1) (2).

Material And Method (2)

Mouse Immunization

Two weeks before collecting the dual-immunization bleed serum (ISR, standing for immunized bleed serum after re-immunization), a mouse was bled to collect the pre-immunization serum (PS). Then, the mouse was injected with commercial sheep red blood cells (SRBC), without adjuvants, into the peritoneal cavity, about 0.5 mL in volume, to elicit an immune response. After one week, the bleeding for the first immunization serum (IR, standing for immunized serum) was performed, and serum obtained. Then, a second injection was immediately performed with the same sheep red blood cell suspension as before. After one more week, ISR was collected by bleeding the mouse for the second time. All bleed sera were stored at at 4°C.

Hemagglutination  Array Loading

0.1 mL of SRBC was added to 1 mL phosphate buffer saline PBS (pH 7.4) solution for washing to remove the formalin preservation components of the commercial product. The washing column was centrifuged at 500 g force for 5 minutes; then, the pallet was resuspended in 1 mL PBS with 0.1% bovine serum albumin (BSA), forming a 10% SRBC suspension.

The 2 collected bleed sera samples were incubated at 56 °C for 30 minutes to denature and inactivate the innate immune complement system proteins (so that the complement system does not lyse cells). Two rows of the 12-column hemagglutination array were loaded with 25 μL of the 10% SRBC suspension. Then, all wells were loaded with their content in Table 1 in PBS solution (pH 7.4) with a volume of 100 μL per well. The negative control group, labeled PBS, did not contain any serum. All other wells contained serum. 

Table 1. Hemagglutination Array Loading Content (2)

Legend: PBS, phosphate-buffered saline; PS, pre-immunization serum; IS, immunized bleed serum; ISR, re-immunized bleed serum diluted; PC, positive control commercial mouse anti-sheep red blood cell serum; 1:10, 1:100, 1:1000 denote dilution factor 10, 100, and 1000, respectively

The hemagglutination array was then incubated and stored at 4°C for 24 hours.

Ouchternoly Plate Loading

A petri dish about 2.5 cm in diameter with 1.2% agarose gel was drilled with a transfer pipet to make 3 wells, as shown in Figure 1. The center well contained rabbit immunoglobulin G (IgG), A (IgA), M (IgM), D (IgD) and E (IgE) antigens. The upper left contained goat anti-rabbit IgG, IgM, and IgA antibodies, and the upper right contained goat anti-rabbit IgG antibodies. The bottom well contained PBS as a negative control. Each well was added 20 μL of their respective content in 2 rounds, 10 μL each round.

Figure 1. Ouchternoly Plate Loading Diagram (2)

Caption: Anti-A, commercial goat anti-rabbit antibodies mixture of IgG, IgM, and IgA; Anti-B, commercial goat anti-rabbit antibody IgG; Rabbit IgG, IgA, IgM, IgD, and IgE at center as antigens; PBS, phosphate buffer saline at pH 7.4

Data And Result (3)

The 0.1% BSA in PBS solution was calculated as the following,

V1C1=V2C2 , V1=V2*C2/C1 = 1.25 mL * 0.1% / 1 % = 1.25 mL * 0.1

V1 = 1.25 mL * 0.1 = 0.125 mL * 1000 μL / 1 mL = 1.25 μL,

1.25 mL - 0.125 mL = 1.125 mL

So, 125 μL of 1% BSA in PBS solution was added to 1.125 mL PBS to form 1.25 mL.

The PS 1:10 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 200 μL = 22 μL , 220 μL - 22 μL = 198 μL

So, 22 μL of Initial PS was added to 198 μL of PBS to form 220 μL.

The IS 1:10 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 250 μL = 25 μL , 250 μL - 25 μL = 225 μL

So, 25 μL of Initial IS was added to 225 μL of PBS to form 250 μL.

The IS 1:100 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 250 μL = 25 μL , 250 μL - 25 μL = 225 μL

So, 25 μL of IS:10 was added to 225 μL of PBS to form 250 μL.

The IS 1:1000 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 220 μL = 22 μL , 220 μL - 22 μL = 198 μL

So, 22 μL of IS:100 was added to 198 μL of PBS to form 220 μL.

The ISR 1:10 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 250 μL = 25 μL , 250 μL - 25 μL = 225 μL

So, 25 μL of Initial ISR was added to 225 μL of PBS to form 250 μL.

The ISR 1:100 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 250 μL = 25 μL , 250 μL - 25 μL = 225 μL

So, 25 μL of ISR:10 was added to 225 μL of PBS to form 250 μL.

The ISR 1:1000 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 220 μL = 22 μL , 220 μL - 22 μL = 198 μL

So, 22 μL of ISR:100 was added to 198 μL of PBS to form 220 μL.

The PC 1:10 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 250 μL = 25 μL , 250 μL - 25 μL = 225 μL

So, 25 μL of Initial PC was added to 225 μL of PBS to form 250 μL.

The PC 1:100 dilution was calculated as the following,

V1 = C2/C1 * V2 , C2/C1 = 1/10, V1= 1/10 * 220 μL = 22 μL , 220 μL - 22 μL = 198 μL

So, 22 μL of PC:10 was added to 198 μL of PBS to form 220 μL.

Figure 2 below shows a photograph of the microtiter 24 hours after the reaction started with antibody serums were added. The agglutination rating (in blue fonts) is based on literature description (2), namely, “4+” for compact granular, “3+” for a smooth mat with folded edges, “2+” for a smooth mat with rough edges, “+/-” for a ring with central precipitation, and “-” for solid central precipitation.

Figure 2. Hemagglutination Array With Agglutination Rating (Gibson, J.)

Caption: PBS, phosphate-buffered saline; PS, pre-immunization serum; IS, immunized bleed serum; ISR, re-immunized bleed serum diluted; PC, positive control commercial mouse anti-sheep red blood cell serum; 1:10, 1:100, 1:1000 denote dilution factor 10, 100, and 1000, respectively. Blue numbers are agglutination ratings.

As shown in Figure 2 with hemagglutination ratings, PBS wells as negative control had negative ratings for agglutination; PS wells as test subjects also resulted in negative reaction ratings. The positive control PC wells had positive ratings between 3+ and 4+, and so did the test subjects with immunizations, IS, and ISR, with positive reaction ratings between 2+ and 4+. For IS, the dilution factor of 100 or higher rendered negative reaction ratings; for ISR, the dilution factor of 1000 or higher rendered negative reaction ratings. So, the titer for the agglutination assay for IS is 10; the titer for the agglutination assay for ISR is 100.

Overall, the titer for IS was 10; the titer for ISR was 100; the titer for PC was 100.

Figure 3 below shows a photograph of the Ouchternoly double diffusion after 5 days of incubation at 4°C.

Figure 3. Hemagglutination Array With Serum (3)

Caption: PBS, phosphate-buffered saline; Anti-A, commercial goat anti-rabbit antibodies mixture of IgG, IgM, and IgA; Anti-B, commercial goat anti-rabbit antibody IgG; Rabbit IgG, IgA, IgM, IgD, and IgE at center as antigens; PBS, phosphate buffer saline at pH 7.4

Discussion

The 2 collected bleed sera samples incubated at 56 °C for 30 minutes indeed denatured and inactivated the innate immune complement system proteins, so the hemagglutination’s negative control groups did not see lysed cells, which would likely have manifested as read cloudly mat.

As shown in Figure 2 with hemagglutination ratings, IS’s titer 10 is lower than ISR’s titer  100. This was per textbook; the first exposure induced IgM production more than IgG, but both in small quantities; the second exposure induced a large IgG production in the follicles of secondary lymph nodes that surpassed the first exposure’s IgM production by a large margin (1). So, the ISR had a higher concentration of antibodies, such as IgG, and it could withstand higher dilution than IS.

Also in Figure 2, the prozone effect of hemagglutination was observed between control group PC 1:10 and PC 1:100, which received reaction ratings of 3+ and 4+, respectively. According to the literature, this was an expected phenomenon, as hemagglutination is highest when an antigen and its antibody are roughly at equal concentrations (2). When antibody concentration was overly high, such as PC 1:10 in the experiment, the agglutination was expected and observed to be lower than when antigen and its antibody were roughly at equal concentrations, as in PC 1:100. There were no prozone effects in IS nor ISR wells. Both IS and ISR wells had the highest agglutination rating when the wells had the highest antibody concentrations. This means that IS 1:10 or ISR 1:100 wells’ antibody concentrations weren’t excessively higher than antigen concentration on the surface of sheep blood cells in the reagent serum.

As shown in Figure 3, the diffusions of antigens and antibodies created precipitations at the curves of equal concentrations. As shown in Figure 4’s illustration below, for the multiple bands of spurs from the IgG+IgM+IgA well (Anti-A well) on the left side, the curves should have straight lines where the diffusion distances of antibodies equal the diffusions distances adjusted by molecular weights of IgM and IgA. These diffusion distances correlate to the triangle geometry pattern, as illustrated in Figure 4 below.

Figure 4. Diffusion distances correlate to triangle geometry (Gibson, J.)

Caption: The 3 pairs of triangle edges correlate to the equal travel distance, pair-wise when adjusted by molecular weights of antibodies and antigens.

The experimental result in Figure 3 showed spur lines as straight lines, as the triangle geometry pattern expects.

On the other hand, as shown in Figure 3’s center curve, the pure IgG well on the right side (Anti-B) released IgG at an equal distance to the IgG released from the left side well (Anti-A). The six arrows in Figure 6’s illustration have equal diffusion distances pair-wise, and the precipitation curve should roughly be symmetrical (when adjusted by the molecular weight of antibodies and antigens) with no spurs.

Figure 5. Diffusion distances create a symmetrical curve by equal diffusion distance (Gibson, J)

Caption: The 3 pairs of triangle edges correlate to the equal travel distance, pair-wise when adjusted by molecular weights of antibodies and antigens.

Indeed, the experimental result in Figure 3 showed a smooth curve similar to Figure 5 between Anti-A and Anti-B, as explained by the equal diffusion distance geometry pattern.

Conclusion

This experiment successfully demonstrated the hemagglutination properties of sera following immunization exposures of non-soluble antigen sheep red blood cells by a mouse. The titer assay showed a higher titer of 100 with a booster shot (second immunization). Without the booster exposure, the tiger was lower, at 10. This was per textbook; the first exposure induced IgM production more than IgG, but both in small quantities; the second exposure induced a large IgG production in the follicles of secondary lymph nodes that surpassed the first exposure’s IgM production by a large margin (1).

The prozone effect in the assay was in line with the literature description (2).

The Ouchternoly double diffusion patterns, with spurs and a smooth curve, occurred as expected. The relationship between the antibodies in the Anti-A well and the Anti-B well was partial-identity, as tested in the experiment. Multiple spurs formed from the Anti-A well, which contained IgM, IgG, IgM, and IgG. Indeed, the Anti-A well had a superset of antibodies of the Anti-B well’s pure IgG content. 

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.