Generation of a Human Monoclonal Antibody to Cross-Reactive Material 197 (CRM197) and Development of a Sandwich ELISA for CRM197 Conjugate Vaccines원문보기
Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune resp...
Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.
Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
To develop a sandwich ELISA for detection of CRM197 in biological samples, mouse anti-CRM197 polyclonal antibodies were produced by immunizing three mice with the purified CRM197, and each antiserum (#1-#3) was serially diluted (1:5,000–1:100,000 v/v) to perform an indirect ELISA.
To examine whether 3F9 can bind to CRM197 conjugate vaccines, one type of Vi-CRM197 and five types of PnPSCRM197 conjugates (serotypes 9V, 11A, 18C, 19A, and 19F) were prepared and purified (Fig. 4), as described in the Materials and Methods, and their reactivity to 3F9 was measured by sandwich ELISA. As shown in Fig.
The 3F9 bound to CRM197 and CRM197-polysaccharide antigen conjugates tested. To our knowledge, this study is the first to show the generation of human mAb to CRM197 and its application in a sandwich ELISA for CRM197 conjugate vaccines. This ELISA system will be useful for quantification and characterization of CRM197 and CRM197 conjugate vaccines.
대상 데이터
To analyze individual anti-CRM197 Fab clones, 285 colonies were randomly selected and phage Fabs were screened by indirect ELISA and quantitative ELISA using BSA. Forty clones with the highest antigen-binding activities were selected and 13 unique clones were obtained after DNA sequencing (Fig.
이론/모형
To convert the selected Fabs into IgG format, the cloning and expression of IgG were performed as described previously [34]. The IgG was purified from the culture supernatant by affinity chromatography on Protein A-agarose beads, and the protein concentration was determined using a NANO-DROP 2000 (Thermo Fisher Scientific). The integrity and purity of the purified antibody were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
성능/효과
Taken together, the results indicate that 3F9 antibody binds specifically to a linear epitope in the B fragment of CRM197 with a high affinity. Considering the facts that CRM197 contains the single amino acid substitution from glycine to glutamic acid at position 52 in the A fragment and that 3F9 binds to a linear epitope in the B fragment, 3F9 may bind to the B fragment of native diphtheria toxin. It remains to be elucidated whether this antibody may have neutralizing activity against the toxin.
In conclusion, we generated a human mAb (3F9) that binds specifically to the B fragment of CRM197 with a high affinity and developed a sandwich ELISA using the human mAb as the capture antibody and mouse anti-CRM197 polyclonal antibody as the detection antibody. The 3F9 bound to CRM197 and CRM197-polysaccharide antigen conjugates tested.
It was found that CRM197 was an ideal carrier for conjugate vaccines against encapsulated pathogenic bacteria such as Haemophilus influenzae type b (Hib), Streptococcus pneumonia, and Neisseria meningitides serogroups A, C, W-135, and Y[9-14]. The use of carrier protein greatly enhances the immunogenicity of polysaccharide or oligosaccharide antigens, which are poorly immunogenic in the immature immune systems of infants and young children [13, 15-19].
후속연구
1D). Finally, four unique Fab clones (3F9, 1E1, 1F3, and 2A9) with the highest antigen-binding activities were selected for further study. The HCDR3 of 3F9 has 13 amino acid residues, while that of the other clones has 8 residues, which are different from each other.
참고문헌 (35)
Galazka AM, Robertson SE. 1995. Diphtheria: changing patterns in the developing world and the industrialized world. Eur. J. Epidemiol. 11: 107-117.
Porro M, Saletti M, Nencioni L, Tagliaferri L, Marsili I. 1980. Immunogenic correlation between cross-reacting material (CRM197) produced by a mutant of Corynebacterium diphtheriae and diphtheria toxoid. J. Infect. Dis. 142: 716-724.
Giannini G, Rappuoli R, Ratti G. 1984. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 12: 4063-4069.
Bigio M, Rossi R, Nucci D, Antoni G, Rappuoli R, Ratti G. 1987. Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies. FEBS Lett. 218: 271-276.
Leonard EG, Canaday DH, Harding CV, Schreiber JR. 2003. Antigen processing of the heptavalent pneumococcal conjugate vaccine carrier protein CRM(197) differs depending on the serotype of the attached polysaccharide. Infect. Immun. 71: 4186-4189.
Kelly DF, Snape MD, Clutterbuck EA, Green S, Snowden C, Diggle L, et al. 2006. CRM197-conjugated serogroup C meningococcal capsular polysaccharide, but not the native polysaccharide, induces persistent antigen-specific memory B cells. Blood 108: 2642-2647.
Usonis V, Bakasenas V, Lockhart S, Baker S, Gruber W, Laudat F. 2008. A clinical trial examining the effect of increased total CRM(197) carrier protein dose on the antibody response to Haemophilus influenzae type b CRM(197) conjugate vaccine. Vaccine 26: 4602-4607.
Avery OT, Goebel WF. 1931. Chemo-Immunological Studies on Conjugated Carbohydrate-Proteins : V. The Immunological Specifity of an Antigen Prepared by Combining the Capsular Polysaccharide of Type Iii Pneumococcus with Foreign Protein. J. Exp. Med. 54: 437-447.
Broker M, Berti F, Schneider J, Vojtek I. 2017. Polysaccharide conjugate vaccine protein carriers as a "neglected valency" -Potential and limitations. Vaccine 35: 3286-3294.
Gerdes J, Schwab U, Lemke H, Stein H. 1983. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer 31: 13-20.
Jung SJ, Seo ES, Yun SI, Minh BN, Jin SD, Ryu HJ, et al. 2011. Purification of capsular polysaccharide produced by Streptococcus pneumoniae serotype 19A. J. Microbiol Biotechnol. 21: 734-738.
Jin Z, Chu C, Robbins JB, Schneerson R. 2003. Preparation and characterization of group A meningococcal capsular polysaccharide conjugates and evaluation of their immunogenicity in mice. Infect. Immun. 71: 5115-5120.
Kothari S, Kothari N, Kim JA, Lee E, Yoon YK, An SJ, et al. 2013. A novel method for purification of Vi capsular polysaccharide produced by Salmonella enterica subspecies enterica serovar Typhi. Vaccine 31: 4714-4719.
Micoli F, Rondini S, Pisoni I, Proietti D, Berti F, Costantino P, et al. 2011. Vi-CRM197 as a new conjugate vaccine against Salmonella Typhi. Vaccine 29: 712-720.
Jo G, Jeong MS, Wi J, Kim DH, Kim S, Kim D, et al. 2018. Generation and characterization of a neutralizing human monoclonal antibody to hepatitis B virus preS1 from phagedisplayed human synthetic Fab library. J. Microbiol. Biotechnol. 28: 1376-1383.
Yoon J-Y, Kim D-H, Kim S, Kim D, Jo G, Shin M-S, et al. 2017. Generation of a monoclonal antibody that has reduced binding activity to VX-inactivated butyrylcholinesterase (BuChE) compared to BuChE by phage display. Biotechnol. Bioprocess Eng. 22: 114-119.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.