Preparation of PrP-specific polyclonal antibody by immunization of PRNP knockout mice with recombinant human PrP protein*_Reference Network (2023)

YANG Xue Hua, WU Yue Zhang, XIAO Kang, GAO Li Ping, CHEN Dong Dong, DONG Xiao Ping,2,3,4,# i SHI Qi,#

1. National Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (Zhejiang University), Institute of Viral Diseases, Chinese Center for Disease Control and Prevention, Beijing 102206; 2. Global Center for Public Health, China Center for Disease Control and Prevention, Beijing 102206 3. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 4. China Academy of Chinese Medical Sciences, Beijing 100700, China

Summary Aim The diagnosis of prion diseases in humans and animals consists in the detection of specific pathological changes in the brain tissue and/or PrPSc in suspected cases. Therefore, the development of methods for obtaining PrP antibodies with good specificity and sensitivity is the basis for prion identification. Methods We generated a PrP-specific polyclonal antibody (pAb P54) in a PRNP knockout mouse model by immunization with full length recombinant human PrP protein residues. We have since validated the pAb in Western blot, immunohistochemistry (IHC) and immunofluorescence (IFA) assays. PrPSc in the brains of experimental scrapie-infected rodents and humans infected with various types of prion diseases. The electrophoretic pattern of brain PrPC and PrPSc observed after reaction with pAb P54 was almost identical to that produced by the commercial monoclonal antibody PrP. The three glycosylated PrP molecules in the brain homogenate were clearly demonstrated by the reaction of these molecules with pAb P54. IHC assays revealed significant PrP deposition in GdnCl-treated brain sections from 139A-infected mice and 263K-infected hamsters. The IFA with pAb P54 also showed a clear green signal surrounding the blue-stained nuclei. and experimental rodent prion diseases.

Key words: prion disease, PrP, antibody, PRNP knockout mice

to introduce

Prion diseases or transmissible spongiform encephalopathies affect a variety of animals and humans. In humans, related disorders include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker syndrome (GSS), and Kuru. Approximately 85% of CJD cases are sporadic (sporadic CJD, sCJD), 10–15% are inherited (hereditary Creutzfeldt-Jakob disease, gCJD), and less than 1% are infectious (iatrogenic Creutzfeldt-Jakob disease) [1,2 ]. ]. Major prion diseases in animals include scrapie in goats and sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer [3].

PrP is a relatively conserved cell membrane protein, expressed mainly in the central nervous system [4,5]. Under the influence of various factors, the normal physiological conformation of PrP (PrPC) is transformed into the abnormal pathological isoform of PrPSc, which is believed to be the etiology of prion diseases [6]. Although the peptide sequences of PrPC and PrPScare are identical, PrPSc has unique biochemical features due to conformational changes, including partial resistance to proteinase K digestion and partial tolerance to GdnCl treatment.

Given the highly conserved nature of PrP, inducing specific antibodies against PrP in wild-type animals is difficult. Most of the available PrP monoclonal antibodies (mAbs) have been generated by immunizing PRNP knockout mice with the PrP antigen [7]. In this report, we generated PrP-specific polyclonal antibodies (pAbs) in PRNP knockout mice by immunization with recombinant human PrP protein. We then verified that the prepared pAbs showed reliable reactivity to PrPC and PrPSc by Western blot detection, immunohistochemistry (IHC) and immunofluorescence (IFA). Our results show that pAb reactivity is comparable to commonly used commercial anti-PrP mAbs.

Materials and methods

Expression and purification of proteins

The recombinant plasmid p-Hu-PrP23-231 was constructed to express the full-length human PrP protein from aa 23 to 231 (rHuPrP23-231), and the expressed rHuPrP23-231 was purified in E. coli as previously described [8].

animal immunity

Transgenic mice (Tg-PrP0/0) knockout of native PRNP [9] were housed in the SPF laboratory. Then, 50 μg of purified rHuPrP23-231 was injected into the hind leg muscles of mice for immunization. About 15 days later, the same amount of rHuPr-P23-231 was injected intramuscularly for reinforcement. Tail blood samples from the immunized mice were collected 15 days later and antibody titers were measured by an established PrP-coated ELISA technique. If the resulting antibody titer was less than 1:20,480, the mice were re-immunized. Mice were sacrificed under anesthesia, blood was drawn and aliquoted.

PrP coated indirect ELISA

An intermediate ELISA protocol was established to test the specificity and sensitivity of the prepared antibodies. Briefly, rHuPrP23-231 was diluted in phosphate buffered saline (PBS) to a final concentration of 1 μg/ml and coated in the wells of a 96-well microtiter plate overnight at 4°C. After washing with PBST (phosphate buffered saline, pH 7.6, containing 0.05% Tween-20), the plates were blocked with 2% BSA in PBST at 37°C for 2 hours and then incubated with various dilutions of serum samples. After extensive washing with PBST, the captured antibody was further reacted with horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody (Jackson, USA) diluted 1:10,000 for 1 hour at 37°C. The color of the solution was developed with 3,3',5,5'-tetramethylbenzidine (Sigma, USA) at 37°C for 30 min. The absorbance was measured at 450 nm (Δ450 nm) after the wells had expired by adding 2 mol/L H2SO4.

Brain homogenate preparation

To evaluate the proposed PrP antibody, homogenates were made from stored brain tissue from experimental rodents infected with sheep scrapie and from humans infected with various prion diseases. Samples included postmortem brain tissue from 1 sCJD case, 1 G114V gCJD case, and 3 FFI cases; brain tissue from mice infected with scrapie agents 139A, ME7 and S15 and hamsters infected with scrapie agent 263K. The clinical and neuropathological features of human and experimental rodent cases have been described in an earlier paper [10]. As controls, a brain from a person who died in a car accident and brains from normal mice and aged hamsters were also included. Brain tissue was washed three times with Tris-buffered saline (TBS, 10 mmol/L Tris-HCl, 133 mmol/L NaCl, pH 7.4) and then washed with lysis buffer (100 mmol/L NaCl, 10 mmol/L EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 10 mmol/l Tris, pH 7.4) containing a cocktail of group III protease inhibitors. The homogenate was centrifuged at 2000 × g for 10 minutes, and the supernatant fraction was aliquoted and stored at −80 °C for further experiments.

Cell culture

The SMB-S15 cell line and the accompanying normal SMB-PS cell line were obtained from the Roslin Institute, UK. PrPSreplication is maintained in this cell line along with cell passage. SMB-PS was established from SMB-S15 cells that were completely prion-free by treatment with pentane sulfate, with no detectable PrPSc. SMB cells were maintained at 33°C in a humidified 5% CO 2 atmosphere in Dulbecco's modified Eagle's medium (VS500T, Auabian, Australia) containing 10% fetal bovine serum.

Western blot

Brain homogenate aliquots were separated by 12% SDS-PAGE and electrotransferred onto nylon membranes. Membranes were blocked with 5% skim milk in TBS for 2 hours at room temperature (RT), then incubated with pAb P54 (1:1000) or commercial PrP mAb 6D11 (1:2000) or 3F4 (1:5000) at 4°C C overnight. After washing with TBS containing 0.1% Tween 20, the membrane was incubated with HRP-conjugated secondary antibody (goat anti-mouse secondary antibody-HRP-Ab, 115-035-003, 1:2000) for 1 hour at room temperature. The blots were developed using an enhanced chemiluminescence system (ECL, PerkinElmer, NEL103E001EA) and visualized on autoradiographic film (General Electric). Images were acquired with ChemiDocTMMXRS + Imager and quantified with Image J software.

Rodent brain homogenates were digested to a final concentration of 50 μg/ml and human and cellular proteinase K to a final concentration of 20 μg/ml at 37°C before Western blot analysis 1 hour to detect the presence of proteinase K resistant PrPSc.

immunochemia

Brain tissue was fixed in 10% buffered formalin and paraffin sections (5 μm thick) were routinely prepared [11]. Sections were quenched with 3% H2O2 in methanol for 10 minutes to endogenous peroxidases and blocked with 5% BSA for 15 minutes at room temperature. Incubate sections with pAb P54 (1:100) or commercial anti-PrP mAb (1:200) overnight at 4°C. The sections were then incubated with an HRP-conjugated secondary antibody (ZSGB-BIO, PV-6002) for 1 hour at 37°C and incubated with 3,3-diaminobenzidine tetrahydrochloride (Boster, AR 1000). Incubate for observation. Sections were counterstained with hematoxylin (Boster, AR 0005), dehydrated and mounted in Permount. Finally, the brain sections were exposed to a buffer containing 4 mol/L GdnCl for 1 hour at room temperature, followed by routine IHC testing to detect PrPSc.

Immunofluorescence analysis

The IFA study was performed according to the previously described protocol [11]. Briefly, brain sections were treated with 0.3% Triton-X100 for 30 minutes, blocked with 5% BSA for 1 hour at room temperature, and then incubated with prepared pAb P54 (1:100) or a commercially available anti-PrP antibody (6D11 , 1:200, Santa Cruz, Sc-58581) overnight at 4°C. The sections were then incubated with goat anti-mouse antibody conjugated to Alexa Fluor® 568 (1:200, Thermo, A-11004) for 1 hour at 37°C. After staining with 1 μg/ml 4′6-diamidino-2-phenylindole (Beyotime, China) for 30 min, sections were placed in Permount and observed using an Operetta (PerkinElmer) or Olympus FV1000 confocal microscope.

ethical statement

Immunization of experimental mice (Tg-PrP0/0) with rHuPrP23-231, preparation of PrP-specific antibodies, prion diseases in humans, and use of experimental rodent brain samples in this study were validated in this study. Ethics Committee of the Institute for Prevention and Control of Viral Diseases, China Center for Disease Control and Prevention, protocol number 2009ZX10004-101. In addition, all animal housing and experimental protocols were in compliance with Chinese regulations for the management of laboratory animals.

result

Expression and immunization of recombinant human PrP

Full-length recombinant human PrP protein amino acids 23-231 (rHuPrP23-231) was expressed and purified from E. coli. coli. SDS-PAGE revealed a band of approximately 21 kD that immunoreacted positively with PrP-specific mAbs 6D11 and 3F4 in western blot analysis (Figure 1). Tg-PrP0/0 mice, 4-8 weeks of age, were immunized intramuscularly with 50 μg of purified rHuPrP23-231 and boosted every 2 weeks until serum titers exceeded 1:20,480 as determined by PrP-coated ELISA. Mouse blood was then collected, serum isolated and pooled as pAb P54.

Reactive titers of pAb P54 by ELISA

The reactive titers of the prepared antibodies were assessed by indirect ELISA on aliquots of pooled pAb P54 sera using plates coated with rHuPrP23-231. Pre-immune mouse serum was used as a negative control. As shown in Figure 2, the negative serum A450nm value was 0.368 at a 1:100 dilution and gradually decreased to less than 0.2 at a 1:1280 dilution. In contrast, the A450nm pAb P54 was 2.758 at 1:100 dilution, peaked at 2.945 at 1:200 dilution and decreased to 0.416 at 1:20480 dilution. Given that a ratio of A450nm test sample/A450nm negative control ≥ 2.1 is considered a positive reaction, the pAb P54 ELISA titer was estimated to be 1:20,480.

The reactivity of pAb P54 was determined by Western blotting

Mouse, hamster and human brain homogenates were prepared separately and examined by Western blotting to evaluate the reactivity of the prepared pAb P54 with native PrP in normal brain tissue. Upon reaction with pAb P54, three typical PrP bands were detected in the brain homogenate at about 25-30 kD, representing double, single and non-glycosylated PrPC (Figure 3A). The blot also showed a pattern similar to the reaction of the homogenates with the PrP-specific mAbs 6D11 (Figure 3B) and 3F4 (Figure 3C). mAb 6D11 responds to PrPC in mouse and hamster brain homogenates, but elicits a very weak signal in human brain homogenates. In contrast, mAb 3F4 did not recognize PrPCin in mice, but responded well to hamsters and humans. In addition, no PrP bands were observed in the brain homogenates of Tg-PrP0/0 mice treated with pAb P54 or mAb 6D11 or 3F4.

Brain homogenates from mice and hamsters infected with different scrapie drugs, as well as human brains infected with different types of prion diseases, were prepared to test the ability of pAb P54 to recognize abnormal PrPSc. After digestion with proteinase K, three 21-30 kD migrators were detected in the brain homogenate spots of mice infected with scrapie agents 139A, ME7 and S15 treated with pAb P54 (Fig. 4A, left) and mAb 6D11. team (correct). In contrast, no PrP signal was detected in the control homogenate patches. A PK-resistant PrP band similar to that seen in mAb 3F4 (Fig. 4B, right), left, was also detected in Western blots of brain homogenates of hamsters infected with scrapie 263K strain of sheep treated with pAb P54 (Fig. 4B, left). These results strongly suggest that the prepared pAb P54 can recognize pathological PrPSc in the brains of scrapie-infected experimental rodents.

Brain tissue homogenates from various human prion disease infections, including 1 sCJD, 1 G114V gCJD and 3 FFI cases, were exposed to pAb P54 and detected by Western blotting. Similar to the reaction with mAb 3F4 (Fig. 5, lower panel), Western blot of pAb P54 (upper panel) revealed PK resistance bands in sCJD and G114V gCJD brains and a weak signal of PK resistance in FFI-3 brains. These findings demonstrate that the prepared pAb P54 is capable of detecting PrPScin of various types of human prion diseases.

Determination of pAb P54 reactivity by immunohistochemistry

Brain sections of normal and 139A-infected mice as well as normal and 263K-infected hamsters were prepared and subjected to a PrP-specific IHC assay to test pAb P54 reactivity; here mAb 6D11 was used as a control. Brain sections were treated with 4 mol/L GdnCl prior to incubation with a single PrP-specific antibody to remove PrPC from brain tissue. Brown PrP signals of varying intensity, indicating positive reactivity, were distributed both inside and outside the brain tissue cells of 263K-infected hamsters (Fig. 6A) and 139A and ME7-infected mice (Fig. 6B); vacuole. In contrast, no obvious brown signal was detected in brain slices from healthy animals. pAb P54 produced images of PrPSc deposits similar to those produced by mAb 6D11 in mouse and hamster brains.

The reactivity of pAb P54 was determined by immunofluorescence assay

The feasibility of the prepared pAb P54 was tested by IFA. A light green signal was noted in SMB-S15 and SMB-PS cells treated with pAb P54 (Figure 7A). In contrast to the green signal generated by mAb 6D11, which is mainly located on the cell surface, the signal generated by pAb P54 is distributed in the cytoplasm and on the cell surface. When brain sections of normal mice and hamsters were analyzed, a bright green signal surrounding blue-stained nuclei was observed in sections reacted with pAb P54. In addition, p54 pAb produced a morphological pattern similar to mAb 6D11 in mouse sections (Figure 7B) and mAb 3F4 in hamster sections (Figure 7C).

discuss

In this report, we generated PrP-specific pAb P54 by immunizing PRNP knockout mice with recombinant human full-length PrP protein. ELISA, Western blot and IHC data confirmed that the newly prepared antibodies can react with recombinant PrP protein, normal brain PrPC in healthy rodents and humans, and pathological PrPS in experimental rodents infected with various scrapie drugs, including 139A, ME7, S15, and 263K, and people infected with various types of prion diseases, including sCJD, G114V gCJD, and FFI. The western blot of pAb P54 clearly showed bands for the three glycosylated forms of PrP, whether PrPC or PrPSc, and these bands showed an electrophoretic pattern similar to that produced by commonly used commercial PrP mAbs. This result indicates that pAb P54 not only has good PrP specificity, but also has the ability to recognize different PrP molecules in a broad spectrum.

Our pAb P54 showed broad reactivity with PrP molecules of various species, including humans and experimental rodents; The protein reaction is minimal. Although PrP proteins are quite conserved across animal species, some PrP-specific mAbs have shown species limitations [12,13]. Many pAbs show broad reactivity with related antigens from different sources and with different epitopes [14], sometimes making them better for capturing more relevant antigens in the sample under study. The presence of PK-resistant PrPScin brain lysates by Western blot and the presence of PrPSc deposits in IHC sections treated with PK or GdnCl are the main criteria for the final diagnosis of human prion diseases [15,16]. The excellent performance of the prepared pAb P54 demonstrated in this Western blot and IHC study reflects its potential use in identifying PrPscin in the brains of humans and experimental rodents infected with prion diseases.

As a product of host genes, the PrP protein is generally conserved in mammals [17,18]. Therefore, eliciting a superior immune response simply by administering the PrP protein to wild-type animals has often been difficult. PRNP knockout animals are an ideal basis for generating PrP-specific pAbs. In this study, we used a strain of PRNP knockout mice with no detectable PrPC expression in their brains to generate pAb; this strategy represents one approach that can overcome the difficulties described above. The prepared pAb P54 showed good specificity and acceptable sensitivity in recognizing native brain tissue PrPC and PrPScin. Another limitation of pAb production for immunization of PRNP knockout mice is the limited amount of blood present in the animals, meaning that a large number of mice are required to obtain sufficient amounts of antibodies. Individual differences in immunized mice are inevitable. To address this issue, we separately assessed the serum titers of individual mice during the second immunization by ELISA and only those mice with a titer greater than 1:20,480 were sacrificed. The prepared pAb P54 was immunized with mouse serum without further purification. Although pAbs have relatively lower reactive titers than mAbs, the simple preparation procedure presented in this paper makes pAb P54 an economical alternative to its monoclonal counterpart. Further purification and concentration of pAb P54 will help to increase its immunoreactivity titer.

Date of receipt: 2019-12-18;

Accepted: June 15, 2020