BIOTECHNOLOGY TECHNIQUES Volume 10 No.5 (May 1996) ~~367-370 Received as revised 22nd March.

LONG-TERM PRESERVATION OF ANTIBODY ACTIVITY AND BINDING TO POLYESTER CLOTH BY DESSICATION

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

Carleton University Ottawa, Ontario, Canada, KlS 5B6

SUMMARY

During 70 days of dessicated storage at 32°C over CaSO,, rabbit IgG and rabbit anti- horseradish peroxidase antibody remained adsorbed onto polyester cloth, retaining full immunoactivity both as an antibody and an antigen. After dessicated storage, the adsorbed antibody could not be released from the polyester cloth by agitated washing in any of the following anhydrous water-mixable organic solvents: methanol, glacial acetic acid, dimethyl sulfoxide, dimethylformamide, or 1,4-dioxane.

INTRODUCTION

We have developed a cloth-based enzyme immunoassay (CEIA) that uses a hydrophobic macroporous polyester cloth as a support for enzyme immunoassays (EIA) (Blais and Yamazaki, 1989a). CEIA has several advantages over commonly used microwell- based EIA. The rate of reaction in CEIA is faster than in microwell EIA because of the large surface area of the cloth as well as shorter diffusional distances between samples and immobilized immunoreactants. The macroporosity of the cloth permits faster washings. Multiple samples applied onto a cloth sheet can be simultaneously processed by washing or saturating the sheet with EIA reagents, thereby eliminating the need for numerous pipettings as required in microwell EIA. In addition, antibody coated polyester cloth may be used to immunoconcentrate dilute antigen from large sample volumes that would go undetected using traditional EIA methods (Blais and Yamazaki, 1989b; Boyd and Yamazaki, 1995a).

The stability of antibody coated polyester cloth at ambient temperatures permits shipment of CEIA kits as well as use in the field without the need for refrigeration. We have previously shown that antibody adsorbed onto polyester cloth can be stored for at least 70 days without any loss of immunoactivity at 3, 3”C under air-dried conditions exposed to uncontrolled ambient humidity (Boyd and Yamazaki, 1995b). Since ambient humidity may greatly vary under field testing conditions, storage under dessicated conditions may render reproducible stability of antibody coated cloth independent of ambient humidity. The present study describes the effects of dessication on the activity and stability of antibody adsorbed onto polyester cloth.

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MATERIALS AND METHODS

Materials The following materials were obtained from Sigma: goat anti-rabbit IgG antibody-

horseradish peroxidase conjugate (anti-rabbit IgG HRP conjugate) (A-61 54), goat anti-rabbit IgG antibody-biotin conjugate (biotinylated anti-rabbit IgG) (B-7389), rabbit anti-horseradish peroxidase antibody (anti-HRP) (P-7899), rabbit IgG (I-5006), avidin-horseradish peroxidase conjugate (avidin-HRP conjugate) (A-3 15 l), horseradish peroxidase (HRP) (P-6782), 3,3’,5,5’-tetramethylbenzidine (TMB) (T-2885), and benzalkonium chloride (B-1383). BioRad supplied the non-fat skim milk protein blocker (protein blocker) (170-6404). The drierite dessicant (CaSO,), 8 mesh regular, was acquired from BDH (B27625). DuPont supplied the polyester cloth (Sontara 8100).

Effect of Dessicated Storage on the Antigen or Antibody Activity of Antibody Adsorbed to Polyester Cloth

Segments (6 mm x 6 mm square) of polyester cloth were soaked in ethanol for a few minutes, washed five times with PBS (0.01 M sodium phosphate pH 7.3 buffer in 0.85 % NaCl), and blotted. Each cloth segment was incubated with 50 ,LL~ of rabbit IgG (50 ng in PBS/cloth) or anti-HRP (100 ng in PBS/cloth) overnight at 32 “C in closed Petri plates. The cloths were then washed five times with PBST (PBS containing 0.05 % Tween 20), blotted, allowed to air-dry for one hour, and then stored at 32 “C in a vacuum sealed dessicator over the drierite dessicant, CaSO,. At regular time intervals over 70 days, cloth segments were removed and assayed for immunoactivity. After incubating the cloth segments in 0.5 % (w/v) protein blocker in PBS for one hour at room temperature (RT), the cloths were washed five times with PBS and blotted. To measure antibody activity, each rabbit IgG cloth or each anti-HRP cloth was incubated with 25 ,LL~ of anti-rabbit IgG HRP conjugate solution (diluted 1:500 in PBS) or 25 ,LL~ of HRP (50 ng in PBS) respectively for 20 min at RT. The cloths were then washed five times with PBST, blotted, and assayed for HRP activity. The amount of anti-rabbit IgG HRP conjugate or HRP adsorbed onto control cloths (coated only with protein blocker) were subtracted from the above results.

Effect of Various Anhydrous Solvents Upon the Adsorption of Antibody to Polyester Cloth Polyester cloth segments (6 mm x 6 mm square) were soaked in ethanol, and then

washed with PBS and blotted as above. Each cloth segment was treated with 50 ~1 of biotinylated anti-rabbit IgG (50 ng in PBS/cloth) and incubated overnight in closed petri plates at 32°C. After washing the cloths five times with PBST, blotting, and air-drying for one hour, the cloth segments were stored in a vacuum sealed dessicator over drierite for five days at 32°C. The cloths were then washed three times while agitating (five min per washing, 2 ml/cloth segment) with PBS or one of the following anhydrous solvents: methanol, glacial acetic acid, dimethyl sulfoxide, dimethylformamide, or 1 ,Cdioxane. After washing three times with PBS and blotting, the cloths were mixed in 0.5 % (w/v) protein blocker in PBS for one hour at RT. Each cloth was then washed three times with PBS, blotted, and incubated with 25 ,LL~ of avidin-HRP (50 ng in PBS/cloth) for 20 min at RT. The cloths were then washed five times with PBST, blotted, and assayed for HRP activity. The amount of avidin-HRP adsorbed onto control cloths (coated only with protein blocker) were subtracted from the above results.

-Assay of HRP activitv One ml of TMB indicator system was added to an individual cloth segment placed

in a 16 mm x 100 mm test tube and shaken at 180 RPM (on a New Brunswick Gyrotary

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shaker) for 10 min at 30°C. The peroxidase reaction was stopped by adding 250 ~1 of 2 N H,SO, and then the absorbance of the resulting colour was measured at 450 nm. The TMB indicator system was prepared by mixing 2.5 ml of TMB (2 mg/ml of ethanol) with a solution consisting of 0.21 g citric acidH,O, 0.42 g EDTA.4 Na, 0.03 g NaBOiH,O, and 100 ml H,O.

RESULTS AND DISCUSSION

Effect of Dessicated Storage on the Antigen or Antibody Activity of Antibody Adsorbed to Polyester Cloth

Limited amounts of rabbit IgG and anti-HI@ antibody were adsorbed onto segments of hydrophobic polyester cloth so that any loss of immunoactivity could be detected. The cloth segments were then stored at 32°C in a vacuum sealed dessicator over CaSO,. The antigen activity of rabbit IgG and the antibody activity of anti-HRP antibody during 70 days of dessicated storage were measured periodically by reaction with excess anti-rabbit IgG HRP conjugate or HRP.

1: Table Effect of dessicated storage on the antigen or antibody activity of rabbit antibody adsorbed to polyester cloth”

“Rabbit IgG or anti-HRP adsorbed onto polyester cloth segments was stored at 32°C in a vacuum dessicator over CaSO, for 70 days. Cloth segment samples in 8 replicates were periodically removed and assayed for antigen or antibody activity as described in Methods. The data (n=S) represent the HRP activity (mean absorbance at 450,, f standard deviation) of anti-rabbit IgG HRF’ conjugate reacted with rabbit IgG cloth or HRP reacted with anti-HRP cloth, respectively. Non-specific adsorption of HRP activity on the control cloths were all below 0.0 15.

Table 1 shows that both antigen and antibody activity of rabbit antibody adsorbed onto polyester cloth were stable during 70 days of dessicated storage at 32°C. Therefore, there was very little denaturation and/or desorption of the rabbit antibody from the cloths demonstrating that antibody polyester cloth can be shipped and stored under dessicated conditions in the absence of refrigeration for use in CEIA. While antibodies were used in the present study, it should be possible to similarly design CEIAs that are useful for detecting antibodies in samples using adsorbed polypeptide antigens.

Effect of Various Anhvdrous Solvents Utlon the Adsorption of Antibody to Polyester Cloth The structure of polyester (Fig. 1) suggests that adsorption of antibodies is primarily

a result of hydrophobic interactions with polyester aromatic groups and hydrogen bonding with polyester c.arbonyl oxygen atoms. It is generally accepted that hydrophobic interactions requires the presence of water for their preservation.

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HO

t

CH,-CH2-0-C - OH

Figure 1: The structure of polyester.

Since antibodies remained adsorbed onto polyester cloth during dessicated storage (Table l), we wanted to know the strength of antibody adsorption in anhydrous water-mixable organic solvents. Biotinylated anti-rabbit IgG was adsorbed onto polyester cloth and vacuum dessicated over CaSO, for five days at 32°C. The antibody cloths were washed three times by agitating in one of the following anhydrous organic solvents: methanol, glacial acetic acid, dimethyl sulfoxide, dimethylformamide, or 1,4-dioxane. The amount of antibody remaining on the cloth was determined by reaction with excess avidin-HRP followed by HRP assay. None of these washings released any of the antibody from the cloth. Agitated washing with these anhydrous water-mixable solvents must have reduced water activity to a minimum. In addition, some of these solvents (e.g. glacial acetic acid and dimethylformamide) are known to be effective in disrupting hydrogen bonds. Thus, antibody adsorption, if maintained by hydrophobic interactions, is very stable even in a minimum of water activity (caused by dessicated storage and water extraction with the solvents).

ACKNOWLEDGEMENTS

This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to Hiroshi Yamazaki and an NSERC Postgraduate Scholarship to Scott Boyd.

REFERENCES

Blais, B.W. and Yamazaki, H. (1989a). Biotschn. Techn., 3,23-26. Blais, B.W. and Yamazaki, H. (1989b). inf. 3: Food Microbial., 9, 63-71. Boyd, S. and Yamazaki, H. (1995a). Biofechn. Techn., 9, 849-852. Boyd, S. and Yamazaki, H. (1995b). Imnunol. Invest., 24, 795-803.

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