BIOTECHNOLOGY TECHNIQUES Volume 7 No.11 (November 1993) p.827-832 Received 30th September

EFFICIENT AND GENTLE ELUTION OF ANTIBODIES FROM AN IMMOBILIZED POLYPEPTIDE ANTIGEN

BY SATURATED MAGNESIUM CHLORIDE

Scott Boyd and Hiroshi Yamazaki' Department of Biology and Institute of Biochemistry

Carleton University Ottawa, Canada KlS 5B6

Amodelantigen, rabbit immunoglobulin G, was immobilized onto polyester cloth by adsorption. The antigen cloth was reacted with sheep anti-rabbit IgG antibody. Antibody bound to the antigen cloth was nearly quantitatively eluted by saturated MgCl,, whereas a commercial antibody eluent slowly eluted only about 70 % of the antibody. Exposure of antibody to saturated MgC12 for 30 min resulted in no loss of immunoactivity. Saturated MgCl*, therefore, is an ideal eluent in immunoaffinity purification of antibodies.

INTRODUCTION

Immunoaffinity chromatography provides a high degree of immunoreactant (antibodies and antigens) purification through a simple procedure of adsorption and elution. However, elution of tightly adsorbed immunoreactants generally requires harsh conditions which often causes denaturation of the eluted or immobilized immunoreactants. Further, the elutionmay not be quantitative when high affinity antibodies are involved. Thus, the development of gentle and efficient elution conditions is desired for immunoaffinity purification. High concentrations of salts such as MgCl, are considered to be gentle agents for dissociating antibody- antigen interactions (Johnstone and Thorpe, 1982). Although 3.5 M MgCl, is listed as an eluent (Harlow and Lane, 1988; Hermenson et al., 1992), its effectiveness has not been sufficiently documented. This paper demonstrates that a saturated MgCl, solution nearly quantitatively desorbs polyclonal antibodies from an immobilized polypeptide antigen.

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Materials The followingmaterialswere obtained from Sigma Chemical Co.:

Sheep anti-rabbit IgG antibody biotin conjugate (biotinylated antibody) (B-9140), streptavidin-HRP (S-5512), rabbit IgG (I-5006) and3,3',5,5'-tetramethylbenzidine(TMB) (T-2885). BioRadsupplied the Blocker (non-fatmilk) (170-6404) and BDH supplied themagnesium chloride (MgC12) (B29096). The ImmunoPure Gentle Ag/Ab Elution Buffer (21013) was obtained from Pierce.

Preparation of antibody-antigen cloths Segments (6 mm square) of polyester cloth (DuPont, Sontara

8100) were soaked in ethanol for a few minutes and then washed thoroughly with PBS (0.01 M, pH 7.3 sodium phosphate buffer in 0.85 % NaCl). After blotting the segments, 50 ~1 of rabbit IgG (100 pg/ml in PBS) or PBS alone (control) was added. Following an overnight incubation at30"C, the cloth segmentswerewashed 5 times with PBST (PBS containing 0.05 % Tween 20). The cloths were then mixed in PBS (containing 0.5 % Blocker) for 1 h on a shaker followed bywashing 5 times in PBS and blotting. Rabbit IgG (antigen)-cloths were reacted for 30 min at room temperature with 30 pi/cloth of biotinylated antibody (diluted 1000 times in PBS), washed 5 times with PBST, and blotted.

Assay of cloth-bound biotinylated antibody Antigen cloths treated with biotinylated antibody were

incubated with 30 pi/cloth segment of streptavidin-HRP (1 pg/ml in PBS) for 30 min at room temperature, washed with 5 times PBST, and blotted. Individual cloth segments were then incubated for 10 minwith lml of a TMB indicator system at room temperature. The absorbance of the resulting colour at 370 nm (A370) was measured after the reaction was stopped with 0.1 ml of 0.2 M NaF. The TMB indicator system was prepared by mixing 2.5 ml of TMB (2 mg/ml ethanol) with a solution consisting of 0.21 g citric acid*H,O, 0.42 g EDTA.4 Na, 0.03 g NaB04*4 H20, and 100 ml H20.

Test for desorption of cloth-bound antipen in saturated M&l, Antigen cloths were prepared as above except that a 1 pg/ml

(instead of 100 pg/ml) solution of rabbit IgG was used to coat the cloths. Since coating the cloth with less than 1 pg/ml of rabbit IgG resulted in a corresponding decrease in capturing of the biotinylated antibody, any loss of antigen from the cloth should be detectable. The antigen cloths were shaken in saturated MgC12 or PBS (control) for 30 min at room temperature, followed by washing 5 times with PBST and incubating in PBS containing 0.5 % Blocker for 1 h (1 ml/cloth). The cloths were reacted with biotinylated antibody and assayed for cloth-bound antibody as described above. The loss of antigen from the cloth was measured by the difference in ho between the control and the antigen cloth treated with saturated MgCl,.

Elution of antibody with M&l, solutions Antigen cloths were reacted with biotinylated antibody to

produce antibody-antigen (Ab-Ag) cloths. MgC12 was dissolved in water at various concentrations. Both Ab-Ag cloths and unreacted Ag cloths were shaken in the MgC& solutions (1 ml/cloth segment) or PBS (0 M MgCl,) at room temperature. After 30 min, the liquids were separated from the cloths via filtration. The cloths were washed 5 times with PBST, blotted and then assayed for cloth-bound biotinylated antibody as above. Non-specific binding of streptavidin-HRP conjugatewas determined by using the unreacted Ag cloth. A37,, due to the non-specific binding was subtracted from the A 370 for each sample to obtain antibody-specific binding of the HRP conjugate. The amount of antibody eluted (A370 eluted from cloths) by MgC& was calculated by subtracting A37,, of MgCl,-treated cloths from A370 of PBS-treated cloths. The degree of elution (%) is then

100 X (A370 eluted from cloths)/(A,,, of PBS-treated cloths).

RESULTS AND DISCUSSION

Effect of MgCl, concentrations on antibody elution To study the elution of antibody from an immobilized poly-

peptide antigen matrix, a model antigen, rabbit immunoglobulin G (IgG), was adsorbed to polyester cloth. First, it was confirmed that the adsorbed antigen was not desorbed during exposure to a saturated MgCl, solution (as described in Methods). The antigen clothwas then allowed to react with a biotinylated F(ab'), fragment of sheep antibody to rabbit IgG. The biotin label was used to permit the assay of the cloth-bound antibody through its binding to a streptavidin-HRP conjugate. The F(ab'), fragmentwas used instead of a whole IgG molecule to avoid the complication of non-specific binding of IgG due to denaturation of the Fc region which an eluent may cause. The Fc is much more susceptible to denaturation than F(ab'), which exposes its hydrophobic site causing non-specific adsorption of IgG (Tijseen, 1985).

As an eluent, 3.5 M MgC12 in a pH 7.2 phosphate buffer has been suggested (Harlow and Lane, 1988). However, the MgC12 solution became cloudy and precipitated. The use of other approximately neutral buffers (e.g. 0.2 M citric acid buffer, pH 6.9) also failed to yield clear solutions. Therefore, MgCl, dissolved in water was used for elution of antibody from the antibody-antigen cloths. Fig. 1 shows that the degree of antibody elution increased as MgCl, concentration increased. Although 3.5 M MgC12 eluted less than 70 % of the antibody, saturated MgCl, (approximately 6 M) eluted more than 90 % of the antibody. The antibodies eluted at higher concentrations of MgCl, most likely respresent high-affinity antibodies. In immunoassays, high-affinity antibodies provide greater detectability of antigens than low-affinity antibodies. Thus, nearly quantitative recovery of polyclonal antibody with saturated MgC12 is highly beneficial for immunoassays.

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Figure 1: The effect of MgCl, concentrations on antibod elution. Antibody-antigen cloths were treated wit K various concentrations of MgC12 and the degree of antibody elution was measured as described in Methods. The pE values of the MgCl, solutions in Hz0 are also plotted. The vertical bars represent standard deviations (n=6).

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Figure 2: The effect of varying pH of MgCl, on antibody elution. Antibody-antigen cloths were treated with 3 N MgC12 at various pH values. The degree of antibody elution was measured as described in Methods. The vertical bars represent standard deviations (n=6).

Effect of varying PH of M&l, on antibody elution Fig. 1 shows that higher concentrations of MgClz inwater gave

lower pH values with a pH of 3.6 for saturated MgCl*. Thus, it is conceivable that lower pH values rather than higher concentrations are responsible for the greater elution observed with MgC12 solutions. To test this, 3 M MgCl, inwater was adjusted to various pH values (3 to 6) with NaOH or HCl, and examined for antibody elution as above. Fig. 2 shows that lowering pH from 5 (pH of 3 M MgCl*) to 3.5 results in only a 5 X increase in the degree of elution. Since raising MgCl, concentrations from 3 M to saturation caused more than a 30 % increase in the degree of elution (Fig. l), the concentration of MgCl, is the primary factor affecting antibody elution.

Comparison with a commercial Ab/Ag elution buffer Commercially available are a few brands of antibody elution

buffers which are claimed to provide quantitative recovery of antibody in immunoaffinity chromatography. Fig. 3 compares the performance of one such eluent "ImmunoPure Gentle Ag/Ab Elution Buffer" (Pierce) with saturated MgCl,. The elution by this commercial eluentwas slow and less efficient than saturated MgC12. Even after 4 h shaking, only 73 % of the antibody was eluted with this eluent. With saturated MgCl*, more than 90 % elution occurred within 10 min.

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Figure 3: Comparison of saturated MgCl, with a commercial Ag/Ab elution buffer. Antibody-antigen cloths were shaken in saturated MgCl,, ImmunoPure Gentle Ag/Ab Elution Buffer or PBS (control for no elution) at room temperature. At various times, cloths were removed, washed 5 times with PBST, blotted and assayed for degree of elution as described in Methods. The vertical bars represent standard deviations (n=6).

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Effect of saturated MsCl, on antibody and antigen activitv Saturated MgQ, though efficient in antibody elution, should

not denature.antibody activity during elution. To test this, stock biotinylated antibody was diluted 10 times in saturated MgClz or PBS (untreated control) and incubated for 30 min at room temperature. The solutions were then diluted 100 times with PBS such that the MgCL concentration became so low so as to not inhibit immunoreaction. The antigen cloths were reacted with the diluted antibody and assayed for bound antibody as described in Methods. The amount of bound antibody was the same whether the antibody was treated with MgClz or not. Similarly, antigen cloth was exposed to saturated MgCl, for 30 min at room temperature. There was no significant change in the capacity for the antigen cloth to capture antibody. Therefore, saturated MgClz does not denature antibody or antigen during a 30 min elution period.

This paper has demonstrated that saturated MgCl, nearly quantitatively elutes polyclonal antibodies from a polypeptide antigen matrix without causing the denaturation of antibody activity. Therefore, saturated MgClz is useful as an eluent in immunoaffinity chromatography.

ACNNONIZDEGEMENTS

This work was supported by Natural Sciences and Engineering Research Council of Canada Grants to HY.

REFERENCES

Harlow, E. and Lane, D. (1988). Antibodies - A Laboratory Manual. PP. 547-549, New York: Cold Spring Harbor Laboratory.

Hermanson, G.T., Mallia, A.K. and Smith, P.R. (1992). Immobilized AffinityLiqand Techniques. pp. 333-334, San Diego: Academic Press, Inc.

Johnstone, A. and Thorpe, R. (1982). Immunochemistry in Practice. Oxford: Backwell Scientific Publications.

Tijseen, P. (1985). Practice and Theory of Enzyme Immunoassay. PP. 301-305, Amsterdam: Elsevier.

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