Lysine at position 222 of the goat prion protein inhibits the binding of monoclonal antibody F99/97.6.1

Abstract

Prion protein (PrP) is encoded by the PRNP gene, which is highly polymorphic in goats, with polymorphisms encoding amino acid substitutions at the protein level. In the current study, the reactivity of monoclonal antibody (mAb) F99/97.6.1 in binding PrP from goats polymorphic at PRNP codon 222 was investigated. Nervous tissue from 30 scrapie-negative goats with 3 different genotypes (222Q/Q, 222Q/K, and 222K/K) was analyzed by Western blot using mAbs P4 and F99/97.6.1. Although PrP was detected in all 30 samples by mAb P4, detection of PrP by mAb F99/97.6.1 was limited to 222Q/Q (12/12). No PrP was detected by mAb F99/97.6.1 in the 222K/K samples ( n = 6), and the signal intensity of mAb F99/97.6.1 for PrP was lower for the 222Q/K samples (12/12 samples). To further investigate these results, additional Western blot analyses were performed, and the PrP signals detected by mAbs F99/97.6.1 and SAF84 were then quantified. The mean F99/SAF84 ratio (± standard deviation) calculated for the 222Q/Q group was 0.73 ± 1.26, and the mean for the 222Q/K group was 0.27 ± 1.31. Statistical analysis of these values evidenced statistically significant differences between the 222Q/Q and 222Q/K samples. The results of the study thus revealed an inhibition by lysine at position 222 on the binding of mAb F99/97.6.1 to goat PrP. This has implications for the use of mAb F99/97.6.1 for diagnostic purposes. Because the 222K allele could be a target for genetic selection in goats, the differential reactivity of mAb F99/97.6.1 could be exploited with a genotyping test setup.

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats

“…PRNP polymorphisms might reduce the sensitivity of the assays in goats carrying specific genotypes by reducing antibodybinding epitopes. 18,20 In our samples, however, statistical analysis did not show any association between failure of each test and goat genotypes (data not shown). The reason for this finding remains therefore unknown and might simply depend on low levels of PrP Sc .…”

PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

“…The results of our study (Figs. 1, 3), and others, 24 clearly demonstrate that the presence of a sequence variant at 222 reduces sensitivity of Western blot detection of PrP C by F99. This difference is also anticipated to be observed when analyzing PrP Sc by Western blot, IHC, or other immunoassays that use F99, and could lead to an increased number of false-negative results.…”

“…A 2012 study demonstrated decreased detection of PrP C with F99 by Western blot in goats with at least 1 allele encoding K222 and the complete absence of signal in K222 homozygotes. 24 In our study, we assessed the performance of several antibodies for the detection of PrP C by Western blotting and PrP Sc by IHC when the caprine prion protein was expressed as wild type or several allelic variants that are present in the United States. 37 In addition to F99, our study focused primarily on F89, because this antibody was included in the original third-eyelid testing scheme for sheep 25 ; the aa epitope it recognizes includes sites of allelic variation in caprine PRNP that may be associated with delayed incubation of disease or reduced risk of infection, 4,6,8,13,14 and naturally infected goats with this allelic variation have been identified in the United States.…”

“…1,7,22,38 A 2012 study demonstrated that allelic variation at codon 222 of the normal cellular prion protein (PrP C ) in goats decreases epitope recognition by mAb F99/97.6.1. 24 It is critical that immunoassays for scrapie consider the potential for this or other alterations to become more prevalent in the goat population worldwide if selection schemes are implemented based on observations of reduced incidence or protection against disease. For example, the third-eyelid test in sheep was originally designed to include mAb F89/160.1.5, 25 but restriction to a single epitope may have yielded falsenegative findings because of the presence of an alternative allele in crossbred sheep.…”

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats

“…In a previous study, we demonstrated, using a Western blot method, a total inhibition by lysine at position 222 on the binding of the monoclonal antibody (mAb) F99/97.6.1 to goat PrP C in healthy goat brain and we proposed the ratio between the signal intensity given by the mAbs F99/97.6.1 and SAF84 (referred to simply as F99/SAF84) to discriminate goats carrying the 222Q/Q, 222Q/K and 222K/K PrP genotypes [ 19 ]. In the present study, we applied the same approach to investigate the contribution of the PrP 222K variant to PrP res formation in natural and experimental scrapie positive goats.…”