Block
Wish
Test Sera
Wish
Conjugate
Wish
Substrate
Stop
Absorbance
Coat BSA (100/^g/ml; 100^1/well) on Flow ICN Linbro plates 24 hours 4®C PBS Tween 20 x3 1% OVA in PBS Tween 20 1 hour 37‘^C PBS Tween 20 x3
Standard curve fig-ng (affinity purified specific antibody) mouse sera 1/100, 1/1000, 1/10000
90 minutes at 3TC
PBS Tween 20 x3
Goat anti-mouse IgG (Fc specific) Alkaline phosphatase 90 minutes at 3TC PBS Tween 20 x3 p-nitrophenyl phosphate (PNPP) 30 minutes at 37°C 3M NaOH 410nm
3.3 ELISA for the Detection of OVA in Mouse Serum
Although the intestinal absorption of immunoreactive antigens such as OVA has been widely reported in mammals (Swarbrick et al 1979, Kilshaw and Cant 1984, Husby
et al 1985, Bruce and Ferguson 1986b, Bruce et al 1987), the immunologcal and molecular features of this phenomenon remain poorly understood.
The detection limits of previously reported assays for the measurement of OVA have been 32 ng/ml in the sandwich ELISA system (Bruce and Ferguson 1986b, Bruce et al 1987), 0.3ng/ml in the biotin avidin amplified sandwich ELISA system (Husby et al 1985) and down to 0.05ng/ml in a radioimmunoassay (Swarbrick et al 1979). The levels of circulating OVA in mouse serum after feeding OVA have previously been shown to be 30-50ng/ml, 5 minutes after feeding the protein (Peng et al 1990) and therefore in the present studies the biotin-avidin sandwich ELISA system was developed. This assay was chosen since it would also give faster results and avoid the use of radioactive labels.
3.3.1 Purification of rabbit anti-OVA IgG antibodies
The sandwich OVA ELISA amplified with biotin avidin was modified from the method of Husby et al (1985). In this ELISA system , rabbit anti-OVA IgG antibodies were used as capture antibodies coated to the solid phase and biotinylated rabbit anti-OVA IgG antibodies were used as detector antibodies. Both capture and detector antibodies were affinity purified from rabbit anti-OVA hyperimmune sera. OVA specific antibodies were isolated from the hyperimmune rabbit sera using a sim ilar procedure to that employed for the purification o f mouse anti-OVA antibodies (see section 3 .1 .1 .). First, OVA specific antibodies were isolated by affinity chromatography. The purified anti-OVA IgG antibodies were then selected from the antibody mixture by binding to protein G as previously described (3.1.1). The activity of the purified rabbit anti-OVA IgG was determined by a chequerboard ELISA. Briefly the plate was coated with OVA overnight at 4°C. Serial dilutions of
antibody were then added and incubated for 90 minutes at 37°C. After washing, 100/xl aliquots of a 1/1000 dilution of sheep anti-rabbit IgG conjugated to HRPO were added and the plate incubated for 1 hour at 37 °C. After adding OPD (0.5mg/ml) the absorbance was read at 490nm.
2mg o f the purified rabbit anti-OVA IgG antibodies was then biotinylated as described in Chapter 2 (section 2.6) and the resulting biotinylated antibody retained for use as the detection antibody in the ELISA. The activity of the biotinylated antibody was determined by a simple antigen capture ELISA. Briefly, the wells of a plate were coated with OVA (lOO^g/ml; 100^1/well) overnight at 4®C. After washing, serial dilutions of the biotinylated antibody were added and the plate incubated for 90 minutes at 37°C. HRPO conjugated streptavidin (Amersham Ltd., England) was added and the plate incubated for 1 hour at 37°C. After adding OPD (0.5mg/ml), the absorbance was read at 490nm.
3.3.2 Development of sandwich ELISA for OVA detection
To determine the optimum concentrations necessary for both the capture and detection of antibodies a chequerboard ELISA was set up using different concentrations of capture and detector antibodies (Figure 3.2 A, B and C). A general scheme for this ELISA is summarised in Table 3.3 and a typical standard curve is shown in Figure 3.3.
Linbro flat bottomed ELISA plates (Flow ICN) were coated with a 1/1000 dilution of affinity purified rabbit anti-OVA IgG (stock concentration 1 mg/ml) in carbonate coating buffer (100/xl/well) and left for 48 hours at 4°C. The plates were washed (PBS/Tween 20) and then blotted dry. 1 % NGS dissolved in PBS Tween 20 was then added (200/Lcl/well) and left for 1 hour at 37°C. The plates were then washed as before and a range of dilutions of OVA (Fraction V, Sigma Chemical Co), in 1% NGS, 0.01% NMS/PBS Tween 20 (100/xl/well) were used in duplicate to give a standard curve over the range 10/xg-lng. The test sera were added in duplicate
(100^1/well) neat, diluted 1/10 and 1/100 in 1% NGS, PBS Tween 20 solution and incubated at 37°C for 3 hours. The plates were then washed as before and a 1/1000 dilution of a biotinylated rabbit anti-OVA was added (lOO/il/well) for 2 hours at 37®C. After washing and drying, a 1/1000 dilution of streptavidin-HRPO was added (100/^1/well) and the plate incubated for 1 hour at 37°C. The plates were then washed 3 times before the addition of substrate solution (lOOftl/well) containing 0.5mg/ml of OPD in 0.05% H^O^/O.IM Citric Acid/ 0.2M Phosphate Buffer. The colour reaction was stopped after incubation at 37°C for 20 minutes by the addition of 25/lc1 of 4M H^SOywell. The optical density was measured at 490nm using an ELISA reader (Titertek multiscan. Flow). The concentrations of ovalbumin in the test sera were obtained by interpolation from the OVA standard curve (see Figure 3.3).
The co-efficients of variation (CVs) of the sandwich ELISA for OVA were determined at concentrations of lOng/ml and lOOng/ml. The inter-assay CVs were calculated for plate to plate variation within a single run of the assay and were 6.2% and 7.9% respectively (n = 4 ); the variations were also calculated for variation between runs on different days, the day to day CVs, and were 10.5% and 9.3% respectively (n = 8).
3.3.3 Comment
No cross reaction between rabbit anti-OVA IgG and mouse serum was found since the background of the sandwich ELISA for OVA in 100% or 10% mouse serum was very low. The detection limit of this ELISA system was 2ng/ml. For neat mouse serum samples this assay could detect concentrations of OVA as low as 3ng/ml. The maximum detection limit for this ELISA was lOOOng/ml. The range of OVA in mouse serum samples ranged from 10^ to 10^ of ng/ml and therefore 3 dilutions were used for each sample, neat, 1/10 and 1/100 in order to determine the dilution of test sample which landed within the working range of the standard curve.