Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.     

Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base.

Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.     

Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).     

The binding of poly(rI) - poly(rC) to human fibroblasts and the induction of interferon.

Human embryonic fibroblasts produce interferon when incubated at 37 degrees C after being treated at 4 degrees C with poly(rI) - poly(rC), either by addition of the double-stranded duplex or by sequential addition of the constitutent single-stranded polynucleotides. Cells which have been incubated with double-stranded poly(rI) - poly(rC) can be prevented from forming interferon by washing the cells with high concentrations of salt, immediately after adsorption of polynucleotides, or by incubation of the cells with single-stranded polynucleotides. The inhibition is probably due to displacement of the inducing molecule from the cell surface. Interferon production by cells treated sequentially with poly(rI) and poly(rC) is not inhibited by either of these treatments and the polynucleotides are not easily displaced from the cell surface.     

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.     

Effect of antibiotics on the formation of specific antibodies.

Immune response to staphylococcal haemolysin and Haemophilus influenzae capsular antigen administered simultaneously with antibiotics was studied in rabbits. In addition to specific humoral antibodies, the quantitative values of IgG, IgA, IgM and C'3 complement were determined. Statistically significant deficiency of immune response was observed in all cases in animals which were given the antigen with the antibiotic in comparison with the controls which were immunized by the antigen alone. Statistically significant differences were also observed in the levels of immunoglobulins (mainly IgG and IgM) in animals which were given antigens simultaneously with antibiotics in comparison with animals which were given antibiotics alone.     

Cross-reactions with Ascaris suum antigens of sera from mice infected with A. suum, Toxocara canis, and Angiostrongylus cantonensis

Ascaris suum larval excretory-secretory (AsES) antigen and larval (AsLA) as well as adult somatic antigen (AsAA) which were thought to be possibly helpful in the diagnosis of visceral larva migrans (VLM) due to A. suum infection were investigated in the present study. Serum taken from mice orally inoculated with approximately 250 embryonated eggs of A. suum or Toxocara canis, or 40 third-stage larvae of Angiostrongylus cantonensis were assessed by enzyme-linked immunosorbent assay (ELISA) using the AsES antigen, AsLA or AsAA at 1, 2, 3, 4, 6 and 8 weeks post infection (WPI). The titer of serum IgG from mice infected with A. suum increased from 1 WPI and a peak at 4 WPI was observed when it reached approximately three times the level of uninfected control mice. Thereafter, it decreased gradually but remained high as found from 6 to 8 WPI. No cross-reactions of heterologous serum IgG against AsES antigen was observed, whereas heterologous serum IgM exhibited significant cross-reactions to AsES antigen. Cross-reactivities to AsLA and AsAA by heterologous serum IgG as well as IgM antibodies were also observed in the trial. Altogether, the AsES antigen apparently seemed to be superior to the other two somatic antigens when used in the diagnosis of A. suum-induced VLM with serum IgG as tested by ELISA. Moreover, it was the first report to test the possibly antigenic cross-reactivity between A. suum and A. cantonensis.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30-45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n=24), Gulu, Uganda (n=20), Bundibugyo, Uganda (n=33), and the Philippines (n=18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections.

Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities to Chlamydia trachomatis serotypes C, D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due to heterotypic antibodies.     

Visualization of Pseudomonas aeruginosa O antigens by using a protein A-dextran-colloidal gold conjugate with both immunoglobulin G and immunoglobulin M monoclonal antibodies.

Two lipopolysaccharide O-antigen-specific monoclonal antibodies, MA1-8 (an immunoglobulin G1 [IgG1]) and MF15-4 (an IgM), were used to localize the O antigen of the lipopolysaccharide of Pseudomonas aeruginosa PAO1. A protein A-dextran-gold conjugate with an average particle diameter of 12.5 nm was used to label bacterial cells treated with MA1-8, while a second antibody (goat anti-mouse IgM) was required before the same probe could interact with cells treated with the IgM antibody MF15-4. Both antibodies resulted in exclusive labeling of the surface of P. aeruginosa PAO1 but not that of an isogenic O-antigen-lacking rough mutant. When the monoclonal antibodies became attached to the cell surface of P. aeruginosa PAO1, resulting in an even coating, the foldings and other topographic details could not be discerned by negative staining. In thin sections of monoclonal-antibody-treated bacteria, a 20- and a 30- to 40-nm thick amorphous layer was observed around the outside of the outer membrane when MA1-8 (IgG) and MF15-4 (IgM) plus goat anti-mouse IgM antibodies were used, respectively. This amorphous layer presumably resulted from the stabilization of the lipopolysaccharide structure by the monoclonal antibodies which prevented the long O-antigen chains from collapsing owing to dehydration.     

Anti-Lewis X IgM and IgG in H. pylori infections in children and adults

A role of autoimmune processes in the pathology of Helicobacter pylori infections has been suggested. The Lewis determinants present in LPS molecule of H. pylori bacteria have been indicated as the cause of antigenic mimicry. In this study, the prevalence of IgM and IgG antibodies to Lewis X antigen in the sera from children and adults, with or without dyspepsia, infected or not infected with H. pylori, seropositive and seronegative for anti-H. pylori IgG were determined immuno-enzymatically (ELISA). Our results revealed that humans may produce anti-Lewis X antibodies, particularly of IgM class, in the absence of H. pylori infection or H. pylori independent dyspepsia. The production of such antibodies, by healthy children who had never been infected with H. pylori suggested that anti-Lewis X antibodies may occur naturally.     

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell‐independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter ( G1 ). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3 . These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L‐lactic acid) or (Leu‐ or Val‐Aib)_n, and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene.

Poly (dG-dC) . poly(dG-dC) was modified by the reaction with N-acetoxy-N-acetyl-2-aminofluorene. The conformations of poly(dG-dC) . poly(dG-dC) and of poly d(G-C)AAF were studied by circular dichroism under various experimental conditions. In 95% ethanol, the two polynucleotides adopt the A-form. In 3.9 M LiCl, the transition B-form-C-form is observed with poly(dG-dC) . poly (dG-dC) but not with poly d(G-C)AAF. In 1 mM phosphate buffer, poly d(G-C)AAF behaves as a mixture of B- and Z-form, the relative percentages depending upon the amounts of modified bases. The percentage of Z-form is decreased by addition of EDTA and is increased by addition of Mg++. Spermine favors the Z-form in modified and unmodified polynucleotides. No defect in the double helix of poly d(G-C)AAF is detected by SI endonuclease.     

Interactions of anti-poly[d(G-br5C)] IgG with synthetic, viral and cellular Z DNAs

Enzymatically synthesized poly[d(G-br5C)] was used to prepare specific polyclonal and monoclonal anti-Z DNA IgGs. The binding specificities of these antibodies were characterized using left-handed polynucleotides with the sequences d(G-x5C)n and d(A-x5C)n.d(G-T)n (mean = aza, methyl, bromo, or iodo). Polyclonal anti-poly[d(G-br5C)] IgG binds the convex surface of the Z helix as evidenced by the strong requirement for a methyl or halogen group at the C5 position of cytosine. Little or no anti-poly[d(G-br5C)] IgG binding occurs to left-handed DNAs carrying a phosphorothioate substitution in the dGpdC bond or an N-5 aza substitution in the cytosine ring. Anti-poly[d(G-br5C)] IgG can stabilize transient Z DNA structures in both polymer families, thereby displacing the equilibrium in solution between the right-and left-handed DNA conformations. Anti-poly[d(G-br5C)] IgG binding sites are found in all tested covalently closed circular natural DNAs (Form I) at their extracted negative superhelical densities, but not in any of the corresponding relaxed Form II or linear Form III DNAs. Binding of anti-poly[d(G-br5-C)] IgG leads to a reduction in the electrophoretic mobility of Form I DNA (e.g. SV40, phi X174, or pBR322) and to the formation of dimers comprised of the bivalent antibody and two supercoiled Form I DNA molecules. The dimers are converted to monomers by DTT treatment. The formation of IgG-DNA complexes is dependent on external conditions (ionic strength, temperature), the properties of the DNA (torsional stress, sequence), and the immunoglobulin (specificity, valency, and concentration). Higher order oligomeric species, indicative of two or more left-handed segments per DNA molecule are formed in reactions of anti-poly[d(G-br5C)] IgG with M13 RF I DNA but not with SV40, pBR322, or phi X174 DNAs. However, oligomers of the latter are generated with other anti-Z DNA IgGs having a broader spectrum of anti-Z DNA reactivity. Conditions which destabilize natural Z sequences in deproteinized supercoiled genomes are: monovalent salt concentrations at or above the 'physiological' range, high temperature, and topological relaxation with DNA gyrase (in the absence of ATP) or with type I topoisomerases. DNA gyrase (plus ATP) catalyses an increase in DNA negative superhelical density which leads to greater anti-Z DNA IgG binding, indicating the formation of additional left-handed regions. Polytene chromosomes of insect larvae bind anti-poly[d(G-br5C)] IgG specifically and stably at Z DNA sites. The distribution of this IgG binding differs in certain regions from that displayed by anti-Z DNA IgG probes with other sequence specificities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence-dependent opening of double-stranded DNA at an electrically charged surface

It has been shown earlier that the DNA double helix is opened due to a prolonged contact of the DNA molecule with the surface of the mercury electrode. At neutral pH, the opening process is relatively slow (around 100 s), and it is limited to potentials close to -1.2 V (against SCE). The opening of the double helix has been explained by strains in the DNA molecule due to strong repulsion of the negatively charged phosphate residues from the electrode surface where the polynucleotide chain is anchored via hydrophobic bases. Interaction of the synthetic ds polynucleotides with alternating nucleotide sequences/poly(dA-dT).poly (dA-dT), poly (dA-dU).poly (dA-dU), poly (dG-dC).poly (dG-dC)/ and homopolymer pairs/poly (dA).poly (dT), poly (rA).poly (rU) and poly (dG).poly (dC)/ with the hanging mercury drop electrode has been studied. Changes in reducibility of the polynucleotides were exploited to indicate opening of the double helix. A marked difference in the behaviour was observed between polynucleotides with alternating nucleotide sequence and homopolymer pairs: opening of the double-helical structures of the former polynucleotides occurs at a very narrow potential range (less than 100 mV) (region U), while with the homopolymer pairs containing A X T or A X U pairs, the width of this region is comparable to that of natural DNA (greater than 200 mV). In contrast to natural DNA, the region U of homopolymer pairs is composed of two distinct phases. No region U was observed with poly (dG).poly (dC). In polynucleotides with alternating nucleotide sequence, the rate of opening of the double helix is strongly dependent on the electrode potential in region U, while in homopolymer pairs, this rate is less potential-dependent. It has been assumed that the difference in the behaviour between homopolymer pairs and polynucleotides with alternating nucleotide sequence is due to differences in absorbability of the two polynucleotide chains in the molecule of a homopolymer pair (resulting from different absorbability of purine and pyrimidine bases) in contrast to equal adsorbability of both chains in a polynucleotide molecule with alternating nucleotide sequence. It has been shown that the mercury electrode is a good model of biological surfaces (e.g. membranes), and that the nucleotide sequence-dependent opening (unwinding) of the DNA double helix at electrically charged surfaces may play an important role in many biological processes.     

Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum]

Localization and characterization of the antigenic components of sparganum which induced IgG and IgM antibodies in the host were studied by immunohistochemical techniques and SDS-PAGE and Western blotting. The antigen recognized by IgG antibody of rats or mice which were immunized by infection or injection of crude extracts of metacestodes of Spirometra erinacei, was located in the parenchyma of sparganum, especially at the cortex and around the calcareous corpuscles. The immunoreaction was demonstrated not only in the encysted fibrous wall of host but around the arterioles or venules in the connective tissue of host. The antigen recognized by IgM antibody of rats or mice was also observed in the parenchyma of sparganum and in the connective tissue of host. By 5-20% gradient SDS-PAGE and EIBT, we detected antigenic components by IgG and IgM antibodies of the rat or mouse immunized by infection or injection of crude extract of spargana. Twenty-three antigenic bands from crude extracts of spargana were recognized by IgG antibody and 15 components by IgM antibody of immunized rats. Out of the bands recognized by IgG and IgM antibodies, 15 were cross-reacted each other. Twenty components of excretory-secretory proteins from spargana were recognized by IgG, and 5 components by IgM antibody of immunized rats. By IgG and IgM antibodies of immunized mice, 16 components of crude extracts were recognized by IgG antibody and 9 components by IgM antibody. Twenty components of excretory-secretory preparation were recognized by IgG antibody and 5 components by IgM antibody. Thirteen components of crude extracts were cross-reacted by IgG antibody of rats and mice.