Hyperimmune Rabbit Antisera Research Articles

Adherence of Vero cytotoxin-producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins.

Escherichia coli of serotype O157:H7 are Vero cytotoxin-producing enteric pathogens that have recently been associated with outbreaks of haemorrhagic colitis, sporadic cases of haemorrhagic colitis and with the haemolytic uraemic syndrome. The organisms demonstrate attaching and effacing binding to the caecum and colon of orally infected gnotobiotic piglets, chickens and infant rabbits. E. coli O157:H7 cells adhere to the surface but do not invade the cytoplasm of human epithelial cell lines in tissue culture. Since outer membranes, lipopolysaccharides and flagella have been identified as bacterial adhesins on other enteric pathogens, we evaluated their roles in the binding of non-fimbriated E. coli O157:H7 to HEp-2 cells. Hyperimmune rabbit antisera were prepared to whole cells, outer membranes and flagella of E. coli O157:H7. The presence of antibody to homologous antigen was confirmed by dot blot immunoassays. Both antisera and purified outer membrane and flagellar antigens were co-incubated with bacteria and HEp-2 cells to quantitate inhibition of bacterial attachment. Adherence of E. coli O157:H7 to tissue culture cells was inhibited by rabbit antisera raised to whole cells (76.0 +/- 5.6% inhibition compared with bacterial adherence in the presence of pre-immune rabbit serum) and outer membranes (69.2 +/- 3.4% inhibition). In contrast, inhibition of bacterial attachment to tissue-culture cells was significantly less when two antisera to H7 flagella were co-incubated with E. coli O157:H7 and HEp-2 cells (12.4 +/- 7.6%; 6.0 +/- 3.5% inhibition). Outer-membrane extracts inhibited adherence to E. coli O157:H7 to HEp-2 cells in a concentration dependent manner whereas isolated flagella and lipopolysaccharide antigens did not inhibit bacterial attachment.(ABSTRACT TRUNCATED AT 250 WORDS)

A prospective assessment of a reverse passive latex agglutination test in the serodiagnosis of systemic candidosis

A reverse passive latex agglutination test is described based on the sensitization of latex particles by a rabbit hyperimmune antiserum raised against a pressate of C. albicans NCTC 3153. The test accurately diagnosed 12 out of the 18 cases subsequently proven by having two positive blood cultures or a postmortem diagnosis if a maximum antigen titre of 1 in 10 is the cut-off point.

Molecular cloning of DNA coding for outer membrane proteins of Haemophilus influenzae type b.

DNA from Haemophilus influenzae type b was cloned in Escherichia coli with a vector lambda gt11 Amp1. Clones producing antigens reactive with hyperimmune rabbit antisera were identified by colony radioimmune assay. A second screening with hyperimmune serum adsorbed with intact H. influenzae type b bacteria was used to identify those clones producing surface-exposed outer membrane proteins. The proteins expressed in E. coli were coupled to Sepharose and used to affinity purify antibodies which were tested for reactivity with outer membrane vesicles. It was found by Western blotting that the clones were producing antigens corresponding to Mr 49,000, 39,500, and 35,000 major outer membrane protein or antigens of H. influenzae type b. Additional clones could be detected by human serum, but their reactivity was not removed when serum was adsorbed with intact bacteria. One of these studied in more detail was found to produce an antigen present in H. influenzae type b lysate but not in outer membrane vesicle preparations.

Immunotherapeutic effect of the lactobacillus vaccine, Solco Trichovac, in trichomoniasis is not mediated by antibodies cross reacting with Trichomonas vaginalis.

According to the producers of the lactobacillus vaccine, Solco Trichovac, its therapeutic effect in trichomoniasis is achieved by antibodies that are induced by the vaccination and cross react with Trichomonas vaginalis. Common antigens of Lactobacillus acidophilus from Solco Trichovac vaccine and T vaginalis were therefore sought by three different seroreactions. Immune serum against Lacidophilus obtained by vaccinating two healthy human volunteers and two rabbits with the original Solco Trichovac vaccine, as well as hyperimmune rabbit antiserum to T vaginalis, were tested with each of the two micro-organisms. No evidence of antigenic similarity between L acidophilus and T vaginalis was obtained with either serum in any of the three serological tests. A non-specific immunostimulatory effect therefore seems to be a more probable explanation of the mode of action of Solco Trichovac vaccine.

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.

Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species

In previous studies with hyperimmune rabbit antisera, we found evidence of serologic cross-reactivity among Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Because of certain biologic and morphologic similarities of these species, attempts were made to determine if this cross-reactivity related to the attachment protein (P1) of M. pneumoniae. Monoclonal and monospecific antibodies against P1 were used to probe proteins of the other species by immunoblotting. One of the P1 monoclonal antibodies was reactive with a smaller protein of M. genitalium; rabbit antiserum raised by immunization with P1 excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was found to react with a similar-sized protein of M. gallisepticum. These preliminary findings suggest antigenic sharing among the species examined; however, limitations of the methods used are discussed.

Proteins associated with human parainfluenza virus type 3.

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein was visualized in place of the 72, 53, and 12K proteins. The 53 and 12K proteins were interpreted to be the two subunits (F1 and F2) of the fusion protein, and the 72K protein was designated as the HN (hemagglutinin-neuraminidase) glycoprotein. The unglycosylated 40K protein represented the viral matrix protein (M). Immunoprecipitation of infected cell lysates with rabbit hyperimmune antiserum against purified virus confirmed the viral origin of these polypeptides.

Ascaris suum: Protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.

Conditions for haemolysis by flaviviruses and characterization of the haemolysin.

The 17D vaccine strain of yellow fever virus (YF 17D) was used to establish the optimal conditions for lysis of chick erythrocytes. Tissue culture-grown, polyethylene glycol-concentrated virus showed peak activity at pH 5.4 in citrate buffer when incubated at 37 degrees C. A further two- to fourfold increase in titre was obtained by pretreatment of the chick erythrocytes with 250 micrograms/ml trypsin. These conditions were also shown to be optimal for Japanese encephalitis (JE), West Nile (WN) and dengue-2 (den2) viruses. The ratio of haemagglutination (HA) titre to haemolysis (HL) titre approximated to unity, suggesting that the two functions are associated with the same molecule although as separable entities since selective inactivation of the HL activity of the virus was accomplished using 60 micrograms/ml trypsin. HL could be demonstrated at neutral pH if the chick erythrocytes were first subjected to treatment with acidic pH buffer. The effect on the virus envelope is thus not the sole contribution of a low pH environment to optimal HL. Hyperimmune rabbit antiserum prepared against purified YF 17D virions inhibited HA and HL if added before agglutination had occurred by the virus but when added after agglutination had taken place it showed specific anti-HL activity. Monoclonal antibodies that inhibited HA (HAI) by YF 17D did not inhibit HL (HLI) activity when applied after agglutination had taken place. Moreover, monoclonal antibodies specific for the 54K glycoprotein of YF virus but without HAI activity also had no effect on HL when added either before or after agglutination. As yet, we have been unable to identify a monoclonal antibody displaying specific anti-HL activity but all those directed against the 54K envelope glycoprotein possessing HAI activity showed HA to be a prerequisite for HL.

An enzyme immunoassay for identification and quantification of infectious murine parvovirus in cultured cells

An enzyme immunoassay was developed to identify and quantify infectious murine parvovirus. The assay was as sensitive as the fluorescent focus test for quantification of virus in clinical material and was less labor-intensive. Viral infectivity could be inactivated with formalin prior to performance of the enzyme immunoassay. Specificity of the reaction product was shown by neutralization of infectivity using a hyperimmune rabbit antiserum to murine parvovirus.

Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribution of serum antibodies to protein antigens.

The occurrence of antibodies to five dietary protein antigens in the sera from 21 healthy adults was investigated by a modified Farr assay. Antibody to ovalbumin (OA) occurred most frequently (90%) whereas only 24% had antibodies to alpha-lactalbumin (ALA). No correlation was noted between the titer of antibodies against bovine serum albumin (BSA) and OA in the single individual. The avidity constants (10(8)-10(9) l/mol) and cross-reactivities against other albumins of anti-BSA antibodies in two human sera were comparable to that of the antibodies in pooled hyperimmune rabbit antiserum. Crossed radioimmunoelectrophoresis showed serum anti-BSA and anti-OA antibodies to be predominantly of the IgG class (13/13, 10/10), occasionally of the IgA- (6/13, 1/10) and rarely of the IgM class (1/13, 0/10). Analysis by radioelectroimmunoassay (rocket immunoelectrophoresis) of the IgG subclass distribution of anti-BSA and anti-OA antibodies showed total absence of IgG3. In contrast, antibodies of the IgG4 subclass were frequently present even in sera with very low levels of total IgG4.

Purification and characterization of GP90, one of the envelope glycoproteins of respiratory syncytial virus.

The large glycoprotein, GP90, of respiratory syncytial virus (RSV) was purified by affinity chromatography using a monoclonal antibody. Hyperimmune rabbit antiserum directed specifically to the purified GP90 neutralized RSV to high titre but did not inhibit fusion of previously infected cells. 125I-labelled GP90 bound rapidly to HEp-2 cells in an unsaturable manner; binding was inhibited by the hyperimmune rabbit antiserum.

Common antigens of Treponema denticola: chemical, physical, and serological characterization.

A sodium deoxycholate-ethanol extractable antigen (DES-Ag) was obtained from four serotypes of Treponema denticola and characterized by chemical, physical, and serological procedures. The cross-reactivity of these antigens was demonstrated by indirect microhemagglutination, immunodiffusion, and immunoelectrophoresis with hyperimmune rabbit antiserum against each of the T. denticola serotypes. Antiserum against two nonoral treponemes, T. phagedenis biotype Reieter and T. pallidum Nichols strain, did not cross-react with the DES-Ag of T. denticola. Chemical analysis of the DES-Ag indicated that proteins (84%) and hexoses (12%) accounted for 96% of the total dry weight of the antigen. Trace amounts of N-acetylglucosamine (0.6%) were also detected. Fatty acids, including palmitic, oleic, and stearic, were identified by gas-liquid chromatography. Polyacrylamide gel electrophoresis of the DES-Ag of each serotype revealed the presence of two bands. The molecular weights of the bands were estimated to be 58,000 and 31,000 by comparing the electrophoretic mobility of the DES-Ag to that of five protein standards of known molecular weights. Although only a single precipitin line was observed by immunodiffusion when each antigen was reacted against its homologous antiserum, two precipitin lines were evident by immunoelectrophoresis. Antiserum against the DES-Ag of T. denticola was shown to agglutinate whole cells of the homologous serotype. Adsorption of this anti-DES-Ag serum with whole cells of T. denticola resulted in the elimination of precipitating antibodies to the DES-Ag by immunodiffusion. It is concluded that the DES-Ag is a component of the outer sheath of T. denticola.

Use of excretory and secretory products from adult female worms to immunize rats and mice against Angiostrongylus cantonensis infection.

To evaluate their protective potential, excretions and secretions of adult female Angiostrongylus cantonensis were prepared from an in vitro culture fluid and injected together with Freund's complete and incomplete adjuvants into rats and mice. Under appropriate conditions, judging from reduced mortality rates and/or prolonged survival periods, these animals were protected against a lethal challenge infection with 3rd-stage larvae. Although both 'immunized' and unimmunized mice died following such a lethal infection, the survival period for the 'immunized' group was significantly longer than that of unimmunized controls (40.8 compared with 26.7 days). On the other hand, the mortality rate of 'immunized' rats was reduced to 25% as compared with 100% in unimmunized controls. There was also evidence of adverse effects on parasite development in these 'immunized' rats. The number of young adult worms recovered from the brain 21 days after challenge infection and both the number and size of mature male and female worms in the lungs 60 days after infection were significantly reduced in the 'immunized' rats. Moreover, the reproductive activity of female worms was depressed, as evident from the output of 1st-stage larvae in the faeces and the number of eggs released during maintenance in vitro. The quantity of proteins released during in vitro maintenance of adult worms in a chemically defined serum-free medium was 0.2--0.5 micrograms/worm/day. The concentrated culture fluid contained at least 7 different antigenic components when analysed with hyperimmune rabbit antiserum to female excretions and secretions (FES) antigen. Polyacrylamide gel electrophoresis in sodium dodecylsulphate showed the presence of 3 major and at least 12 minor protein components with molecular weights varying from 24,000 to 115,000 daltons. Different lines of evidence presented in this study show immunological cross-reactivity among antigens from different developmental stages.

The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies.

Antigenic variants of the A/PR8 (H0N1) and A/Hong Kong/68 (H3N2) strains of influenza virus were isolated after a single passage of these viruses in the presence of monoclonal hybridoma antibodies to the haemagglutinin. Hyperimmune rabbit antisera reacted (in haemagglutination-inhibition tests) to high titre with both wild-type and variant viruses, but the monoclonal antibodies, which reacted with the wild-type virus to titres of the order of 1/10 5 did not react at all (or to very low titre) with the variants that they selected. This suggests that the changes occurring in the monoclonal variants are restricted to a single antigenic site out of many on the haemagglutinin molecule. Amino acid analysis of the soluble tryptic peptides from the haemagglutinin ‘spikes’ of wild-type and variant viruses suggest that the dramatic loss in the ability of the variants to bind the monoclonal antibody used in their selection is associated with a single change in the amino acid sequence of the large haemagglutinin polypeptide, HA 1 . For PR8 virus, eight out of ten variants selected with one monoclonal antibody showed the same sequence change of serine to leucine in the HA 1 polypeptide. The change in the other two variants was not determined. No sequence data on PR8 haemagglutinin are available, so the experiments were continued with a Hong Kong (H3N2) strain where much of the sequence of HA 1 and HA 2 is known. Three different monoclonal hybridoma antibodies to A/Mem/1/71 (H3N2) haemagglutinin were used to select a total of ten variants of this virus. Variants selected with one monoclonal antibody were not recognized by the other two monoclonal antibodies as being different from wild-type virus, suggesting that the three antibodies bound to different sites on the surface of the haemagglutinin molecules. Each of the variants occurred with a frequency of about 1 in 105 in the wild-type virus. One group of our variants selected with H14/A2 monoclonal antibody showed the same antigenic properties and the same sequence change (asparagine to lysine) in the N-terminal half of HA 1 . Of three variants selected with H14/A20, two showed a different change at a locus also in the N-terminal region of HA 1 (a proline was replaced by serine in one variant and by leucine in the other). Of the other three variants (selected with H14/A21 monoclonal antibody) one showed a change in HAX of serine to tyrosine. This change occurred in residue number 37 of cyanogen bromide fragment 2 (CN2). In the other two variants the change in HAX has not been determined, but in these a tryptic peptide comprising residues 49-56 of CN2 was missing. The tryptic peptides of the HA 1 polypeptide, showing changes in the variants selected with monoclonal antibodies, were also found to undergo sequence changes in naturally occurring Hong Kong variants isolated from man. In each case, however, the sequence changes in the monoclonal variants were different from those in the field strains. No changes were found in the HA 2 polypeptide from any of the variants.

Antibodies against <i>Aspergillus fumigatus</i>

Individual antigenic components of <i>Aspergillus fumigatus </i>were identified by crossed immunoelectrophoresis (CIE) using pooled rabbit hyperimmune antiserum. Patients’ sera with enhanced precipitins against <i>A. fumigatus </i>showed antibodies against identical antigenic components in CIE. Identification of the antigenic components by the intermediate gel technique showed high concentrations of antibodies against a small group of antigenic components. No differences were found in the indiviual antigenic components involved in the precipitation reactions between groups of sera with enhanced and normal IgE levels against <i>A. fumigatus. </i>Crossed immunoelectrofocusing was used to determine the isoelectric point of particular components that were identified by CIE.

Antigenic drift in type A influenza virus: Sequence differences in the hemagglutinin of Hong Kong (H3N2) variants selected with monoclonal hybridoma antibodies

Three monoclonal hybridoma antibodies, designated H14/A2, H14/A20, and H14/A21, which appeared to bind to different sites on the hemagglutinin of A/Mem/1/71 (H3N2) virus, were used to select a total of 10 antigenic variants of this virus. The variants occurred with a frequency of about 1 per 100,000 infectious wild-type virus particles and were isolated after a single passage of the virus in the presence of the monoclonal antibody. Hyperimmune rabbit antisera did not distinguish wild-type and variant viruses, but the monoclonal antibodies, which reacted with the wild-type virus to titers of the order of 1 100,000, did not react at all (or to very low titer) with thevariants they selected. This dramatic change in antigenicity appeared to be associated with a single change in the amino acid sequence of the large hemagglutinin polypeptide, HA1. Four variants selected with H14/A2 monoclonal antibod showed the same antigenic properties and the same sequence change (asparagine to lysine) in the N-terminal half of HA1. Of three variants selected with H14/A20 monoclonal antibody, two showed a different change at a locus also in the N-terminal region of HA1 (a proline was replaced by serine in one variant and by leucine in the other). Of the other three variants (selected with H14/A21 monoclonal antibody) one showed a change in HA1 of serine to tyrosine. This change occurred in residue number 37 of cyanogen bromide fragment 2 (CN2). In the other two variants the change in HA1 has not been determined, but in these a tryptic peptide comprising residues 49–56 of CN2 was missing. No changes were found in the HA2 polypeptide from any of the variants.

Comparison of the Antigens Produced by Foamy Virus in a Cytolytic and a Persistent Infection of HEp2 Cells

The antigens from cytolytic infections of HEp2 cells by type I simian foamy virus produced two multicomponent precipitation lines when tested by immunodiffusion with the homologous hyperimmune rabbit antiserum. The antigens obtained from a non-productive infection of MK5 virus in HEp2 cells produced only those precipitation lines which corresponded with the inner lines obtained from the cytolytic infection. Similarly, hyperimmune rabbit antiserum against antigens extracted from the persistent infection lacked the antibody which was responsible for the outer lines of precipitation. Indirect immunofluorescence with acetone-fixed and unfixed cells using the homologous and heterologous sera confirmed the absence of antigens in the persistent infection and showed that an antigen is produced in the persistently infected cells which is either absent or present in very small amounts in cytolytically infected cells. Neutralization experiments and ether treatment suggested that the missing antigens in the persistent infection were the envelope components of foamy virus. It is proposed that the persistent infection has properties in common with some infections by RNA tumour viruses.

Cross-Neutralization of Infectious Mononucleosis and Burkitt Lymphoma Strains of Epstein-Barr Virus with Hyperimmune Rabbit Antisera

Abstract Antisera that neutralize Epstein-Barr virus (EBV) were prepared in rabbits. Neutralization tests were based on inhibition of transformation of normal lymphocytes into cell lines or on inhibition of induction of early antigen. Supernatant culture fluids of human and marmoset lymphoblastoid cell lines productive of virus were the source of antigen. Neutralizing activity was not induced by immunization of rabbits with medium from EBV transformed cell lines that did not release virus. However, neutralizing activity appeared in the sera of rabbits inoculated with supernatant fluid of a producer line from which nearly all the virions had been removed by ultracentrifugation. Rabbit antisera with neutralizing activity reacted with radioiodinated virus purified by velocity sedimentation on a sucrose gradient. Neutralizing capacity of the sera was not removed by absorption with primary human or marmoset leukocytes, by absorption with EBV transformed human B lymphoid cells that were not virus producers, nor by absorption with marmoset lymphoblastoid T cell lines transformed by Herpes saimiri virus. However, absorption of the antisera with EB virus producer lines caused a marked reduction or elimination of neutralizing antibody titer. Cross-neutralization and cross-absorption tests did not indicate the existence of distinct serotypes. Antiserum prepared against the Hawley EBV strain from a patient with mononucleosis neutralized two viruses of Burkitt lymphoma origin, HR-1 and Olare. Antiserum raised against the nontransforming HR-1 strain neutralized the two transforming strains. Absorption of the sera with virus producer cells removed neutralizing activity directed against both homologous and heterologous viruses. The results indicate that EB viruses of diverse origin and diverse biologic properties share antigens, presumably located both on the viral envelope and on the cytoplasmic membrane of producer cell lines, which elicit neutralizing activity.

Mouse protection test for group B Streptococcus type III.

The mouse model has been used previously for the study of protection against challenge with strains of group B Streptococcus types Ia, Ib, and Ic, but investigators have not yet been successful in causing lethal disease when type III strains are inoculated. In this study, six clinical isolates belonging to serotype III were lethal in seven-week-old outbred albino mice; the 50% lethal dose (LD50) for mice tested ranged from 1.8 x 10(4) to 4.6 x 10(6). In contrast, a laboratory prototype strain of serotype III was not lethal. The lethality observed with this protection test depended on both the volume of the inoculum given and the medium in which the streptococci were suspended. LD50 values decreased as the volume of the inoculum, given intraperitoneally, increased, up to a volume of 1.5 ml. Hyperimmune rabbit antiserum to a clinical isolate of type III protected mice against an estimated 90% lethal dose of the isolate. Normal rabbit serum and heterologous antisera, except for one of two antisera to type Ia organisms, were not protective. Absorption of type III antiserum with whole organisms of serotype III or with purified, type III-specific polysaccharide inhibited protection of mice against type III organisms.