The Elisa guidebook

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10.2.2¡ª

Titration of Enzyme-Labeled mAbs

Plates were coated with 50 µL per well of an optimal dilution of the relevant rabbit anti-FMDV serum in carbonate/bicarbonate buffer (1/5000). After incubation overnight at 4¡ãC or at 37¡ãC for 2 h, the plates were washed. Purified 146S and 12S (50 µL) were then added at 2 µg/mL in blocking buffer and incubated for 1 h at 37¡ãC while being rotated. Each of the conjugated mAbs was tested on the relevant virus capture plates by titration in twofold dilution series from 1 to 10 across 11 wells. The conjugates were diluted in 50 µL of blocking buffer as just described and incubated for 1 h at 37¡ãC while being rotated. Plates were then washed and the chromogen/substrate steps followed as in Subheading 10.2.1. Data were obtained relating the activity of the conjugates on dilution to estimate the optimal dilution to be used in the virus quantification studies.

10.2.3¡ª

Titration of Virus Using Dilutions of mAb as Capture and Detecting Reagents

Optimal dilutions of each of the mAbs, as assessed Subheadings 10.2.1. and 10.2.2., were used to coat wells under the same conditions. After washing, known concentrations of 146S, 12S, TTV or DNV were added as twofold dilution series in 50 µL of blocking buffer (beginning at 5 µg/mL). Plates were incubated at 37¡ãC for 1 h. Labeled mAbs were then added at optimal dilutions in 50 µL of blocking buffer. After 1 h of incubation at 37¡ãC, the plates were washed and substrate/chromogen was added, followed by stopping and reading in a spectrophotometer. Different combinations of mAbs were used as capture and detecting reagents. Virus and virus preparations were also captured using the rabbit serum and then detected with the mAb reagents. Guinea pig serum was also used to quantify the antigens when mAbs and rabbit serum had been used as capture reagents.

10.2.4¡ª

Titration of 146S in the Presence of 12S

The effect of adding high concentrations of 12S to a constant amount of 146S was examined using different concentrations of mAb capture reagents. The same systems were also examined using guinea pig polyclonal detecting serum.

10.2.5¡ª

Standardization of 146S Titrations

A standard preparation of known concentration was established using purified 146S virus quantified by UV spectrophotometry. This standard was stored in small volumes at ¨C70¡ãC, and samples were thawed and used once in the assays. The standard was diluted in quadruplicate in 50 µL of blocking buffer

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to produce a standard curve relating the weight of 146S to OD, and the variation at each point was calculated. For the assay of virus contained in tissue culture samples, four different infected tissue culture supernatants were diluted 1/2, 1/4, 1/8 and 1/16 in blocking buffer and then assayed in quadruplicate in 50-µL vol. The weight of the virus in the samples was then calculated for the different dilutions by referring to the curve obtained for the standard 146S titration. The standard 146S acted as a control for each test plate, and examination of the cumulative data from all the test plates allowed the variation of the assay to be determined.

Determinations of the concentrations of the virus from three of the tissue culture samples were made over ten tests on ten separate days.

10.3¡ª Results

Figure 8 shows the titration curves obtained using different dilutions of mAbs and the polyclonal rabbit serum as capture reagents for constant amounts of the various antigens. Captured antigens were detected using polyclonal guinea pig serum/anti-guinea pig conjugate. All the mAbs captured both 146S and 12S, although the plateau maximum OD for 12S was significantly lower than that for 146S, particularly for mAbs B2 and D9. This was also true for the rabbit capture/guinea pig detector system. Neither TTV nor DNV was detected when the B2 and D9 mAbs were used as capture reagents, whereas mAbs C8 and C9 were capable of presenting TTV to the detecting antibody. The polyclonal system detected all antigens, although dilution of the serum was needed to achieve optimal capture. The optimal dilution for each of the mAbs and the polyclonal sera for the capture of 146S are arrowed.

Figure 9 shows the titration curves for each of the mAb-enzyme conjugates where constant amounts of 146S and 12S were captured by the optimal dilution of polyclonal rabbit serum indicated in Fig. 1. All the mAb conjugates were capable of detecting both antigens, the plateau height differences between 12S and 146S resulted from the limiting factors of the polyclonal rabbit serum to capture 12S as explained diagrammatically in Fig. 10. The reduction in plateau height for the titration of the same weight of 12S was also shown in the polyclonal capture/detection system.

Figure 11 shows the titrations of various antigens using optimal dilutions of the same mAbs as both capture and detecting reagents. The rabbit capture/ guinea pig detection system is also included for the same materials assessed. This shows that B2 and D9 detected 146S but not 12S, TTV or DNV; whereas C8 and C9 detected 12S and TTV. The polyclonal system demonstrated that all the antigens were present in the samples and that there were marked effects on the maximum OD obtainable for 12S and DNV, with a small effect on TTV.

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Fig. 8.

Use of mAbs and rabit sera on solid phase to capture various antigenic preparations of FMDV. ↓ = optimal dilution of capture reagents.

Figure 12 shows the effect of measuring two different concentrations of 146S in the presence of a dilution series of 12S. The rabbit/guinea pig system demonstrated that the 12S was titratable. There was no significant effect on the detection of the constant amount of each of the 146S samples in any of the mAb capture/detector systems.

Error bars representing 2 ¡Á SD from the mean OD of each sample are shown.

Figure 13 shows the results of titrating 146S in the presence of a relatively high concentration of 12S. The rabbit/guinea pig system indicates the level of reaction owing to the 12S observed after the 146S is diluted to a level below that of the 12S. No such plateau is observed in the mAb systems.

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Fig. 9.

Titration of mAb enzyme conugates against 146S and 12S captured by rabbit serum. ↓, Optimal dilution to detect maximum amount of 146S virus.

Figure 14 shows a curve of cumulative data obtained on 10 estimations of the standard 146S performed on different plates over 2 wk using the B2 and D9 mAb systems. The variation in results is shown as bars (1 ¡Á SD mean) for each of the titrations (quadruplicate estimates). A line has been drawn through 1.0 OD to highlight the mean and upper and lower limits of the data for each mAb. This represents the most precise area on the standard curve.

The concentration of virus in unknown samples was best estimated when OD values of 0.6¨C1.3 were obtained, corresponding to a range of approx 0.03¨C0.5 µg/mL of virus on the standard curve. Table 18 shows the results for the determinations of the 146S in four infected tissue culture samples. Table 19 shows data for three of the samples assessed at a single dilution over 10 tests.

Figure 15 is a diagrammatic representation illustrating the relationship B) to 12S subunits derived from those particles (Fig. 15C). The relative sizes of the particles are drawn to scale along with an IgG molecule. A side view of the

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Fig. 10.

Consequences of binding virus and subunits via mAbs or polyclonal sera. (A) Relative subunit number to four virus particles. (B,C) Capture of 146S virus particles and subsequent detection presents more antigenic sites that with optimal capture of subunits generated from the same number of particles, thus affecting plateau height maxima. (D) mAbs orientate subunits to present internal epitopes, unlike presentation of captured 146S particles. (E) Detection

of mAb captured subunits with same mAb as used to capture does not work, as relevant epitopes are already bound, unlike detection of epitopes on whole particles in the same system.

12S pentamer (Fig. 15D) illustrates that epitopes are contained internally as well as externally (in common with 146S external sites). The consequences of mAb or polyclonal antibody capture/detection is examined in Fig. 15E and F.

Note in Fig. 10. Figure 10A¨CC that for the same antigenic mass (illustrated as four virus particles), a higher number of subunits are available for capture. However, the ability of the capture system to bind all the units released on

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Fig. 11.

Titration of mAbs as capture and detection systems against dilutions of 146S, 12S, TTV, and DNV.

producing 12S is more limited. This affects the number of antibodies binding as compared to the antigens exposed on virions. Figure 10D illustrates that the binding of capture mAbs to external 12S epitopes in common with 146S has

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Fig. 12.

Measurement of 146S in the presence of a dilution series of 12S using mAb capture and detection system.

the effect of preventing the same mAb binding, thus allowing differentiation of 12S and 146S particles, even though they share the same epitope.

10.4¡ª Discussion

A novel method is described for the detection and quantification of whole 146S particles of FMDV using mAbs as capture and detecting reagents. The

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Fig. 13.

Titration of 146S in the presence of a constant amount of 12S using mAb capture and detection systems.

key to the success of the system relies on the fact that, although the epitopes identified by the mAbs are common to 12S and 146S, they are on the outer capsid surface. The subsequent presentation of the mAb-bound 146S and 12S to the same mAb allows detection of only 146S since the crossreactive epitopes

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Fig. 14.

Cumulative data for titration of 146S using mAb capture and detection systems.

on the 12S particles are orientated toward the plate by interaction with the capture mAb.

The capture of 12S by the mAbs and polyclonal serum was shown through its detection using the polyclonal guinea pig antiserum detector in Fig. 9. Note the different reaction for the same weights of 146S and 12S by all the systems.

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Table 18

Quantification of 146S Virus

from Four Tissue Culture Samples at Different Dilutions

					Weight from mean value read				Concentration in
						from standard			original (µg/mL)b
						curve (µg/mL)a
Sample				OD	SD	Mean	SD	Mean		¡ÀSD
A	1/2		1.51		0.09	>4	¡ª	>4		¡ª
	1/4		1.45		0.09	1.71	0.10	6.84		6.44¨C7.24
	1/8		1.30		0.06	0.83	0.07	6.64		6.08¨C7.20
B	1/2		1.35		0.07	1.01	0.12	2.02		1.78¨C2.26
	1/4		1.29		0.05	0.81	0.05	3.24		3.04¨C3.44
	1/8		1.10		0.04	0.37	0.03	2.96		2.72¨C3.20
C	1/2		1.12		0.04	0.39	0.02	0.78		0.74¨C0.82
	1/4		0.89		0.03	0.20	0.01	0.80		0.76¨C0.84
	1/8		0.76		0.02	0.11	0.01	0.88		0.80¨C0.96
D	1/2		0.11		0.01	0.008	0.004	0.016		0.008¨C0.024
	1/4		0.10		0.01	0.007	0.004	0.028		0.012¨C0.044
aWeights measured using mean value from sample. The variation of standard curve at this
OD was used to establish the standard deviation (SD) for the test sample.
bObtained by multiplying the weights of virus obtained for the mean and ¡ÀSD by dilution factor.
Table 19
Assessment of 146S Virus from Three Tissue Culture Samples
Using 10 Separate Tests
Sample			Dilution		Weight (µg/mL)	SD (µg/mL)		CV(%)a
A				1/16	7.1	0.51		7.1
B				1/8	3.1	0.19		6.1
C				1/8	0.8	0.05		6.1

aCoefficient of variation (%).

In polyclonal capture/detection systems in the sandwich ELISA, there is always a reduction in the plateau height (a constant maximum OD observed for a range of concentrations in which the detecting serum is in excess), since there is both a reduction in the number of 12S particles (antigenic mass) that can be captured from the equivalent weight of 146S owing to physical reasons, and an orientation factor for 12S (similar to that described for the mAb) depending on the exact nature of the polyclonal antibodies and the extent that the capture and detecting antibodies react with internal and external epitopes. The sandwich