Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment

Abstract

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.

Figure 1

SDS–PAGE of crude formalin-treated RNase A (lane 1), individual fractions of formalin-treated RNase A isolated by gel filtration (lanes: 6, monomer; 5, dimer; 4, trimer; 3, tetramer; 2, a mixture of oligomers higher than pentamer), and native RNase A (lane 7).

Figure 2

Results of direct ELISA on native RNase A (curve 1) and RNase A incubated in 10% neutral buffered formalin at a concentration of 4 mg/ml for 1 day (curve 2). Coating was done at 37°C for 1 h.

Figure 3

Results of capture ELISA on native RNase A (curve 1), formalin-treated RNase A (curve 2), and individual fractions of formalin-treated RNase A (curves: 3, monomer; 4, dimer; 5, trimer; 6, tetramer; and 7, mixture of oligomers higher than pentamer). Coating was done at 37°C for 1 h.

Figure 4

SDS–PAGE of native RNase A (lane 1) and RNase A incubated in 10% neutral buffered formalin for 9 days (lane 2) or in 5% neutral buffered formalin for 1 day (lane 4). The latter two samples were then demodified for 4 h in TAE buffer (pH 4) at 65°C (lane 3, 10% sample; lane 5, 5% sample). M, molecular mass markers in kDa.

Figure 5

Results of capture ELISA on native RNase A (curve 1) and RNase A incubated in 5% neutral buffered formalin at a concentration of 1 mg/ml for 1 day (curve 2) and then demodified for 4 h in TAE buffer (pH 4) at 65°C (curve 3). Coating was done at 4°C overnight.

Figure 6

Results of capture ELISA on native RNase A (curve 1) and RNase A (6.5 mg/ml) incubated in 10% neutral buffered formalin for 9 days (curve 2) and then demodified for 4 h in TAE buffer (pH 4) at 65°C (curve 3). Coating was done at 4°C overnight.

Figure 7

SDS–PAGE of RNase samples. Lanes 1–4: RNase A modified in 5% neutral buffered formalin for 1 day (lane 1) and then incubated in TAE buffer (pH 4) for 2 h at 55°C (lane 2), 75°C (lane 3), or 95°C (lane 4). Lanes 5 and 6: unmodified RNase A after incubation in TAE buffer (pH 4) for 2 h at 55°C (lane 5) or 95°C (lane 6). Lanes 7–10: RNase A modified in 10% neutral buffered formalin for 9 days (lane 7) and then incubated in TAE buffer (pH 4) for 2 h at 55°C (lane 8), 75°C (lane 9), or 95°C (lane 10).

Figure 8

Results of capture ELISA detection of native RNase A (curve 1) and RNase A incubated in 5% neutral buffered formalin for 1 day (curve 2) or 10% neutral buffered formalin for 9 days (curve 3), after a 2-h incubation in TAE buffer (pH 4) at different temperatures. Coating was done at 4°C overnight. Capturing was done from solutions containing 0.2 ng of antigen in 200 μl of PBS.

Figure 9

Molecular diagrams of proposed hydrogen bond formation in (a) amino methylol adducts formed by Asn and (b) an Asn cross-linked with a Lys residue.