Section: 802, (Thursday)
Group: 4
Names: John Gibson, Kyle LaPlant
Instructor: S. Royan, PhD
Date: Nov 07, 2024
University Of Massachusetts,
Lowell, Fall 2024
Immunology Lab Report 4: Purifying Immunoglobulin G With Ion Exchange Chromatography
Contributions:
Objective Gibson & LaPlant
Material/Method LaPlant
Data/Result Gibson
Discussion a) through e), Gibson; f), LaPlant
Conclusion Gibson
Objective
Using ammonium phosphate precipitation, dialysis, and ion exchange chromatography, this experiment aimed to purify immunoglobulin G (IgG) from a given whole rabbit serum. Light absorbing property of IgG at 280 nm (A280) was utilized to select chromatography output fractions according to literature (1). The isolated product was examined with goat anti-rabbit IgG antibody in Ouchterlony double diffusion assays to verify the binding and precipitation efficacy.
Material And Methods (2)
Ammonium Sulfate Precipitation of Immunoglobulins and Dialysis
In a 15 mL conical tube, 1.1 mL of whole rabbit serum was diluted with 1.2 mL phosphate-buffered saline (PBS) (pH 7.2). An additional 40 ul of whole rabbit serum was saved and stored at 4° C for later use. Over the course of 5 minutes, 1.8 mL of saturated ammonium sulfate solution(100%) was added to the diluted sera in single drop increments to get an ammonium sulfate concentration of 44 percent. The tube was rotated throughout to provide constant mixing and was kept in an ice bath. The tube was then centrifuged at 1000 g (2,420 rpm for a 6-inch rotor) for 15 minutes. The supernatant was then discarded, and the pellet was redissolved in 2.4 mL of PBS while on ice. Once again, saturated ammonium sulfate solution(100%) was added to the sample in single drop increments for a total of 1.6 mL or a 40 percent ammonium sulfate concentration. The tube was then centrifuged the same as before, and the supernatant was discarded. The pellet was then dissolved in 1.1 mL of degassed 0.02 M Tris solution with hydrochloric acid (Tris-HCl), pH 8.0, and loaded into a semi-permeable cellulose membrane with a 12-14 kDa molecular weight cut-off. The tubing was then tied off, and the solution was dialyzed against 1 liter of 0.02 Tris-HCl, pH 8.0, with the buffer changed 3 times over a week.
Preparation and Washing of Diethylaminoethyl (DEAE) Affi-Gel® Blue Gel Column
With the flow stopped, 5 mL of 0.02 M Tris-HCL, pH 8 with 0.02% NaN3, was added to a 10 mL plastic column. The column was then gently tapped until air bubbles no longer rose from the white filter at the bottom of the column. Then, 5 mL of diethylaminoethyl (DEAE) Affi-Gel® blue gel was added and allowed to settle for ≈10 minutes. The flow stop was then removed, and the column was pre-washed with two bed volumes (≈20 mL) of 0.02 M Tris-HCL, pH 8, with a constant layer kept over the beads to prevent drying. After the last wash was added, the flow rate of the column was recorded, and the pH was measured using litmus testing strips. The stopper was then placed to stop the flow when the buffer reached the minimal amount to cover the beads. The cap was then put on top of the column before being placed into a 50 mL tube and stored at 4o C.
Isolation of IgG by DEAE Affi-Gel® Blue Gel Bifunctional Chromatography
The column was washed with 1.5 bed volumes of 0.02 M Tris-HCL, pH 8, and the flow rate and pH were measured. Then, a glass tube (10 x 75 mm) was filled with 1 mL of buffer, and a line was drawn to mark the level. Then, using the initial tube as a guide, a 1 mL line was drawn on 20 tubes and they were labeled 1-20. After, the volume of the previously dialyzed sample was measured using a sterile 15 mL tube, and 40 ul was removed and stored at 4o C. Once the wash volume reached the gel surface, the column was clamped and the dialyzed sample was added to the column. The flow was then allowed to resume and the first 1 mL was collected and discarded. From there, 1 mL fractions were collected in tubes 1-20, with approximately 3 bed volumes of 0.02 M Tris-HCl being added to the column during the process.
Determination of IgG Concentration by UV Light Absorption
Each tube containing the fractions were vortexed and 100 ul of sample was loaded into a UV transparent 96-well plate per the loading map (Table 1). The absorbance values were then recorded using a SpectraMax plate reader at 280 nm. Using a graph of fraction # vs absorbance at 280 nm, the peak fraction, 50% ascending fraction, and 50% descending fraction were determined.
Table 1: Loading Map for 96-well Plate (2)
Table 1 contains the order in which the samples were loaded onto the 96-well plate for absorbance measuring at 280 nm.
Detection of IgG by Ouchterlony Plate Reaction
Two petri dish plates of roughly 3 cm diameters and 1.2% 5 mL agarose had gel drilled with a transfer pipette, with five holes in each plate per the template (Figure 1). Each well received 20 ul of the corresponding antigen or antiserum sample denoted in the template. The 20 ul volume was added in 10 ul increments, allowing for the first amount to settle into the wells before adding the remaining amount. The plates were left to rest flat on a benchtop for 15 minutes to allow proper liquid absorption into the agarose. Lastly, the Ouchterlony plates were placed in a 10 cm petri dish with a moistened Kim-Wipe and parafilm sealed before being incubated overnight at 37o C.
Figure 1. Template for loading of Ouchterlony Plates (2). The layout shows the placement of the antiserum and antigens on the two Ouchterlony Plates.
Data And Results (3)
Before dialysis, the resuspension of ammonium phosphate serum precipitate had a volume of roughly 1.1 mL; at the end of the one-week dialysis, the antibody serum suspension column in the membrane was 2.0 mL. It took 1.5-bed volume washing (15 mL) with Tris-HCL over the chromatography volume with DEAE Affi-Gel® to establish a 36 mL/hour flow rate, as calculated here from a timed collection of 1 mL fluid in 100 seconds,
(3600 sec / 1 hour) ÷ (100 sec/mL) = 3600 sec/hour × 1 mL / 100 sec = 36 mL/hour .
This flow rate was slower than the initial column setup’s 48 mL/hour one week prior. A Tris-HCl fluid pH of 8 was immediately observed in the chromatography column before adding the dialysis suspension volume.
Table 2 below shows the A280 measurements of each fraction of the chromatography output eluates. The peak absorbance (subtracted by blank background Tri-HCL solution) was 0.124 with tube 5; the 50% peak absorbance during ascending was 0.060 with tube 2; the 50% peak absorbance during descending was 0.061 with tube 11.
Table 2. A280 Of Chromatography Output Fractions (3)
Legend: A through F, rows of the microtiter array; 1 through 12, columns; Tris, Tris buffer with HCl setting pH at 7.4; A@280, absorbance at light wavelength 280nm; Average, average absorbance of the triplicates; Avg-blank, average absorbance of the tube offset by the background Tris solution; pink filling cells denotes Tris background absorbance measurements; orange filling cells denotes key fractions for further testing, including Ouchterlony tests. Crossing-out fields are outliers that are not used in plotting.
Figure 2 below shows A280 values plotted for each chromatography fraction collection tube number. Outlier data points are excluded from the plot. Figure 2’s A280 (subtracted by background solvent absorbance) curve starts low at the first fraction (first mL) of the chromatography output, climbing rapidly to the peak value of 0.124 at the 5th fraction, and then gradually decreases, matching the literature outline of IgG chromatography elution (2). IgG’s concentration was highest at the 5th mL collection fraction.
Figure 2. A280 (subtracted by background absorbance) Vs. Fraction Collection Tube Number (3)
Caption: Orange arrows denote fractions to be further tested in Ouchterlony and other assays.
According to Figure 2, the fraction collection with the highest A280 absorbance was in tube 5; the 50% absorbance of the peak absorbance during ascending was in tube 2; the 50% absorbance of the peak absorbance during descending slope was in tube 11.
Figure 3 below shows the precipitation arcs of column chromatography product fraction at the peak A280. The goat anti-rabbit IgG antibody diffused from the center well toward the 4 outer wells. The product of this experiment, at the peak A280 fraction, diffused from the lower left toward the center and had a precipitation arc, meeting goat anti-rabbit antibody in the middle between P and AB wells. The control well NC had no precipitation spurs or identity arcs with the goat anti-rabbit body in Ouchterlony double diffusion.
Figure 3. Ouchterlony Double Diffusion Of Peak A280 Chromatography Eluate (3)
Caption:
AB, goat anti-rabbit IgG antibody; NC, negative control with Tris
solution with HCl at pH 7.4;
P, chromatography eluate fraction
of tube 5 for peak A280;
A, commercial rabbit IgG antigen;
PD, post-dialysis of serum after
dialysis but before ion exchange
chromatography
Also shown in Figure 3 above, the commercially prepared rabbit IgG antigen also had a precipitation arc between the A and the AB wells. Also shown in Figure 5, the post-dialysis serum also had a precipitation arc between the PA and the AB wells.
Figure 4 below shows the precipitation arcs of column chromatography product fractions at 50% of peak A280 before and after reaching the peak. Again, the goat anti-rabbit IgG antibody diffused from the center well toward the 4 outer wells. The product of this experiment, at the peak A280 fraction, diffused from the upper left toward the center and had a precipitation arc, meeting goat anti-rabbit in the middle between P and AB wells. The product fraction (tube 2) of this experiment, at the 50% absorbance before peak A280, diffused from the lower right DF well toward the center and had a precipitation arc, meeting goat anti-rabbit antibody with a small gap from the DF well. The product fraction (tube 11) of this experiment, at the 50% absorbance past peak A280, diffused from the upper right AF well toward the center and had a faint precipitation arc, meeting goat anti-rabbit antibody with no gap at the AF well.
Figure 4. Ouchterlony Double Diffusion Of 50% A280 Chromatography Eluate (3)
Caption:
AB, goat anti-rabbit IgG antibody;
AF, 50% of maximum absorbance in
the ascending fractions before
maximum A280 absorbance;
DF, 50% of maximum absorbance in
the descending, post maximum
A280;
P, chromatography eluate fraction
of tube 5 for peak A280;
SM, starting material, serum before
ammonium phosphate and before
dialysis treatment
Discussion
Chromatography is a generic term for separating fluid (liquid or gas) components by physical/chemical properties and isolating them, involving a stationary phase and a mobile phase interaction. Chromatography options for protein purification include 1) gel filtration by molecular weights and molecular shapes, 2) affinity filtration by chemical group bindings to stationary phase, and 3) ion exchange by electrical charge attraction force differences (2). Ion exchange methods can be anion exchange, binding negatively charged species and releasing positively charged species, or cation exchange, binding positively charged species and releasing negatively charged species (4).
This experiment aimed to use combined ion exchange and chemical affinity chromatography to isolate the IgG serum component, as albumin is known to have high affinity binding with commercial stationary phase product Cibacron® blue’s F3GA component, and IgG was expected to elute first in a single step (2).
When ion exchange and chemical affinity chromatography are combined in 1 column of chromatography, such as this experiment’s DEAE Affi-Gel® column, it is called bifunctional chromatography.
In this experiment, anion exchange chromatography was used with the positively charged amine group in the diethylaminoethyl (DEAE) component of the stationary phase DEAE Affi-Gel®. The stronger the negatively charged protein species, the slower the molecule species eluted. In this experiment, IgG had an isoelectric point of 7.3, according to literature (2), and IgG was only slightly negatively charged at pH 8 of the chromatography column and did not bind to DEAE Affi-Gel® as strongly as the serum's albumin and other globulin components. In addition, the Cibacron® blue F3GA component of the stationary phase bound albumin; hence, IgG eluted first in a single step. Figure 3 verifies the isolated product IgG as it has an identity arc with commercial rabbit IgG protein. Figure 5 illustrates the bifunctional mechanism in this experiment.
To determine the relative concentration of IgG product, Figure 2’s A280 (subtracted by background solvent absorbance) shows the eluant started low at the first fraction of the chromatography output, climbing rapidly to the peak value of 0.124 at the 5th fraction and then gradually decreased, matching literature outline of IgG chromatography elution (2). This indicates that IgG’s concentration was highest at the 5th mL collection fraction. Furthermore, Ouchterlony double precipitation worked in this experiment by simultaneously diffusing antibodies and antigens from 2 separate gel wells into the gel, according to the principle of equal-concentration precipitation. In Figure 4, the antibody-antigen precipitation line between P and AB has a broader gap toward P than the gap between the precipitation line between DF and AB toward DF. This indicates that the product IgG solution at peak A280 and in the P well had a higher concentration than the DF well’s IgG solution at 50% of peak A280, so P’s diffusion could reach a longer distance while maintaining an equal concentration. This implicated peak A280 fraction eluate (tube 5) with the highest concentration of IgG product.
If albumin were to be further isolated from the used flow column, high salt concentrations, such as 0.5 M potassium thiocyanate (KSCN), could weaken hydrogen binding between the stationary phase's Cibacron® blue F3GA component and albumin (5). Then, the salt solution eluate would contain albumin.
In Figure 3, the identity arc between A (commercially prepared) and P (experiment product) implicated purified IgG in the P well. The arc between the A and P wells appears thick due to the thickness of the gel in the dish, diffusing the IgG content from either well by a vertical front in the thick gel. There were no spur lines of precipitations either from the commercially prepared rabbit IgG from well A or the experimentally prepared IgG from well P. This signifies the high purity of IgG from both A and P wells. Also in Figure 3, the negative control test with Tris-HCL solution wall produced no precipitation, as expected.
Additional information about the contents of each sample can also be derived from interactions of precipitate lines between two samples. The two precipitate lines formed between the antibody-P wells and antibody-A wells, join together to show an arc of identity (Figure 3). This provides further indication that the P fraction contained IgG since it was similar to the commercial sample. Furthermore, when comparing the precipitate lines of antibody-P wells and antibody-PD wells there is another identity arc. This was as expected since the P well was derived from the sample placed in PD, so the IgG in each well was the same. Similarly, the precipitate lines of antibody-P wells and antibody-SM wells form another identity arc (Figure 4). This was also expected since the IgG in the P well was derived from the SM. The identity arc between the P well and AF and DF wells was not determined either due to proximity or lack of a distinct precipitation line. Overall, IgG was obtained in well P, as expected.
In this experiment, ammonium phosphate ions at 40-55% saturation weakened the hydrogen bond between IgG, other protein components, and water molecules. Once IgG molecules were unbound from water molecules, they precipitated due to heavier molecular weight than water and the buffer component Tris. However, without dialysis and chromatography purification steps, the mixture of many of these serum molecule species automatically precipitates in agarose gel without binding to any antibody or antigen. It exists as a circular halo around the SM well of Figure 4.
Conclusion
In conclusion, this experiment was a total success. The negative control in Figure 3, with no precipitation, implicated low contamination in the experiment. The highest IgG concentration was verified by Ouchterlony double diffusion to be obtained in tube 5. The purified product in tube 5 had an expected double diffusion pattern against rabbit antibody. Furthermore the SM well in Figure 4 confirmed that the dialysis procedure eliminated a wide range of impurities in the serum. This experiment successfully isolated IgG from a whole serum.
References
McCombs, Jessica R, and Shawn C Owen. “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry.” The AAPS journal vol. 17,2 (2015): 339-51. doi:10.1208/s12248-014-9710-8
Royan, S.V. & A. Alton. (2024). Immunology Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.
Gibson, John. (2024). Immunology Laboratory Notebook Fall 2024. [Unpublished Immunology Laboratory Notebook]. Department of Biological Sciences, University of Massachusetts, Lowell.
Cummins, Philip M et al. “Ion-Exchange Chromatography: Basic Principles and Application.” Methods in molecular biology (Clifton, N.J.) vol. 1485 (2017): 209-223. doi:10.1007/978-1-4939-6412-3_11
Yavuz, Handan, et al. "Cibacron Blue F3GA incorporated poly (methylmethacrylate) beads for albumin adsorption in batch system." Colloids and Surfaces A: Physicochemical and Engineering Aspects 223.1-3 (2003): 185-193.
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