Native and Non-Native Conformation-Specific Antibodies Directed to the Loop Region of Hen Egg-White Lysozyme

Abstract

Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV‐1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV‐1 Carrier Sera

“…However, the results were somewhat different from those of typical anti-protein antibody responses. The affinity of anti-protein antibodies to derivative peptides is generally 1/100 to 1/1,000 lower than that to protein antigens (14,29). In contrast, the affinity of the monoclonal anti-rp17 antibodies to the derivative peptides was rather 2 to 29 fold higher than that to the immunizing antigen rp17.…”

“…Secondly, our experience in epitope mapping of hen egg-white lysozyme suggested that a peptide of at least 18 amino acid residues is necessary for titrating anti-protein antibodies in the liquid phase (13). Thirdly, secondary structures, including disulfide bridge formation, are thought to be important for the binding affinity of peptides to anti-protein antibodies (14). From these viewpoints, we synthesized 3 p17 peptides (peptide 12-29, peptide 53-87 and peptide 87-115) and used them to titrate almost all the monoclonal anti-rp17 antibodies.…”

Evaluation of the Affinity Measurement of Anti-HIV-1 P17 Monoclonal Antibody by BIAcore

“…On the other hand, the range of the Kas of 12-29 or rp 17 captured by HyHIVl-6 were all 104 to 106, but the range of the Kds of 12-29 were 10"5 to 10"4, and that of rpl7 were 10"4 to 10~3. Compared with the difference in the kinetic affinities when using [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] or rpl7 as ligand versus using HyHIVl-6 as ligand, the formar KAs were higher than the latter KAs except when HyHIV4 was 10' 10 : 10 10 Association Constant for 12-290 1 ) (Tables 3 and 4). …”

“…HyHIVl-6 were all IgGl types, and stained the HIV-1-infected cells by the indirect immunofluo¬ rescence staining, and they inhibited the viral multiplication of the culture supernatant in the range of 0.5 to 1.0 of logioTCID50 when they were added to the HIV-1-infected cells. "·2·16·17' Furthermore, according to the crystal structure of the HIV-1 pl7 reported by Hill et al,"8' P12-29 consists of helix 1 (residues 11-19) except residue 11, strand 1, (19)(20)(21) loop 1, (21)(22)(23)(24)(25)(26)(27) and strand 2, (27-29) and it forms a part of the basic patch that functions as both the protein's membrane binding surface"9·20' and the nuclear localization signal. '4·5' Thus, both HyHIVl-6 and 12-29 were thought to be significant for fur¬ ther study, so we chose them for the present work.…”

Understanding protein non-folding

“…The immunoglobulins obtained against a given protein may be specific for different levels of macromolecule: the primary structure [177, 178], the secondary structure [179], or the tertiary structure [177, 178]. In the latter case, the antigenic determinants may reside on either the neighbouring residues in the chain (loops) [177, 178] or on spatially distant residues [179]. Furthermore, it has been shown that antibodies in the immune serum may possess a high affinity to the internal elements of an antigen [179].…”