Polyclonal Immune Sera Research Articles

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity.

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.

Ebola treatment shows promise

An experimental treatment for Ebola virus infection is effective in mice. Researchers from the CDC, Emory University and the US Army Institute of Infectious Diseases recently reported their results in the Journal of Virology. Polyclonal immune serum was collected from mice that had survived previous exposure to the virus. Researchers then used the immune serum to prevent infection in other mice. Auspiciously, the immune serum was protective when administered either before or after infection. The study authors say the potential use of convalescent-phase or immune serum to treat Ebola deserves further study. Ebola claimed the lives of 224 people in Uganda during the latest outbreak of the disease. AV

Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication.

Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.

Immunological analyses of human papillomavirus capsids

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease. Questions remain, however, concerning the extent of capsid antigenic similarity between closely related virus genotypes. To investigate this issue, we produced VLPs and corresponding polyclonal immune sera from several anogenital HPV types, and examined these reagents in enzyme-linked immunosorbent assays (ELISAs) and in cross-neutralization studies. Despite varying degrees of L1 genetic sequence relatedness, VLPs of each type examined induced high-titer serum polyclonal antibody responses that were entirely genotype-specific. In an in vitro infectivity assay, only cognate VLP antisera were able to neutralize pseudovirions of HPV-16, HPV-18 and HPV-33, with two exceptions: HPV-31 and HPV-45 VLP post-immune sera demonstrated low levels of neutralizing activity against pseudovirions of HPV-33 and HPV-18, respectively. In other experiments, epitopes shared between closely related types were found to be less immunogenic than, and antigenically distinct from, primary type-specific B-cell determinants of the viral capsid. In addition, results from epitope blocking experiments suggested a close correlation between primary type-specific capsid antigenic sites and virion neutralization. These findings support the view that papillomavirus genotypes denote unique viral serotypes, and suggest that a successful vaccine for these viruses will likely require the inclusion of VLPs of each serotype for which protection is desired.

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections.

Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I.

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

Examination of Polyclonal Rabbit Immune Sera against Serovars of Acinetobacter baumannii and Genospecies 3 for Cross-reactions with Reference Strains of other Named/Unnamed Genospecies of Acinetobacter

Polyclonal rabbit immune sera against 38 serovars of Acinetobacter baumannii and genospecies 13 capable of growth at 44 degrees C and against 26 serovars of genospecies 3 and genospecies 13 incapable of growth at 44 degrees C were examined for serological cross-reactivity with reference strains comprising 17 genospecies (among them 6 named species) of Acinetobacter. Checkerboard agglutination tests yielded very few cross-reactions. Specifically, genospecies 17 cross-reacted weakly with A. baumannii serovar 27; strains genospecies 13 (Bouvet), TU 14, and 'close to TU 13' were strongly agglutinated by antiserum against A. baumannii serovar 18. Genospecies 14 (Bouvet) yielded a very weak cross-reaction with genospecies 3 serovar 20, and genospecies TU 13 reacted weakly with anti-genospecies 3 serovar 15 serum. The 'between genospecies 1 and 3' reference strain proved to be A. baumannii serovar 5 as determined with absorption tests.

Human papillomavirus type 11 recombinant L1 capsomeres induce virus-neutralizing antibodies.

The human papillomavirus type 11 (HPV-11) L1 major capsid protein can be trypsinized to generate recombinant capsomeres that retain HPV genotype-restricted capsid antigenicity (M. Li, T. P. Cripe, P. A. Estes, M. K. Lyon, R. C. Rose, and R. L. Garcea, J. Virol. 71:2988-2995, 1997). In the present study, HPV-11 virion-neutralizing monoclonal antibodies H11.F1 and H11.H3, previously characterized as recognizing two distinct HPV-11 capsid-neutralizing antigenic domains (S. W. Ludmerer, D. Benincasa, and G. E. Mark III, J. Virol. 70:4791-4794, 1996), were each found to be highly immunoreactive with trypsin-generated capsomeres in an enzyme-linked immunosorbent assay (ELISA). Capsomeres were used to generate high-titer polyclonal immune sera that demonstrated HPV genotype-restricted reactivity by ELISA. The capsomere antisera were then tested in an in vitro infectivity assay and found to neutralize HPV-11 virions. In this assay, HPV-11 capsomere polyclonal antisera exhibited neutralization titers (10(-5) to 10(-6)) comparable to those obtained with a virion-neutralizing antiserum raised previously against intact HPV-11 VLPs (R. C. Rose, R. C. Reichman, and W. Bonnez, J. Gen. Virol. 75:2075-2079, 1994). These results indicate that highly immunogenic, genotype-restricted HPV capsid-neutralizing antigenic domains are contained entirely within capsomeres. Thus, capsomeres may be viable vaccine candidates for the prevention of HPV disease.

Therapeutic passive vaccination against chronic Lyme disease in mice.

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 microg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 microg OspC-specific Abs) were repeatedly given (4x in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.

Heterogeneity of Lyme disease spirochaetes within individual vector ticks

To determine whether Lyme disease spirochaetes (Borrelia burgdorferi) within vector ticks (lxodes dammini) sampled from enzootic sites comprise single or mixed populations, we compared their reactivity to a polyclonal rabbit immune serum with that to a battery of monoclonal antibodies (mAb) against OspA, OspB and flagellin. More spirochaetes were recognized by the polyclonal antibody than with the mAbs. Spirochaetes from field-sampled ticks reacted poorly to mAbs against OspB. No such differences in reactivity to polyclonal or monoclonal antibodies were observed for the N40 strain of B. burgdorferi from BSK cultures and infected laboratory-reared vector ticks. We conclude that in nature each tick may be infected by an antigenically heterogeneous mixture of spirochaetes.

Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.

Antigenicity, cross-reactivity and surface exposure of the Neisseria meningitidis 37 kDa protein (Fbp)

The 37 kDa iron-repressible protein, Fbp, was purified from two Neisseria meningitidis strains by metal-affinity chromatography and used to obtain mouse monospecific polyclonal immune sera. Dot-blot, immunoblotting and whole cell ELISA results demonstrate that the Fbp is present in all 16 N. meningitidis and four commensal Neisseria species tested, is highly antigenic in mouse when injected in pure form, and shows intra- and inter-species antigenic homogeneity, anti-Fbp antibodies being fully cross-reactive using the techniques mentioned. We also found that Fbp molecules (or parts of them) are surface exposed, in disagreement with the proposed exclusively periplasmic localization, although anti-Fbp antibodies seem unable to block iron uptake or to induce complement-mediated killing of the meningococci. Taken along with the high immunogenicity of the purified protein and the complete cross-reactivity of the antibodies elicited, this suggests that the protective effect of the purified Fbp must be further studied to evaluate its inclusion in future vaccine trials.

Phenotypic and Genotypic Characterization of Clinically Recovered Presumptive Acinetobacter baumannii Isolates which Failed to Grow at 44 °C

Eleven clinical isolates of Acinetobacter, which exhibited an identical biochemical profile compatible with genospecies 3 and failed to grow at 44 °C, were not agglutinated by polyclonal rabbit immune sera against 26 serovars of genospecies 3. Rather, all 11 isolates reacted strongly with antiserum against serovar 18 of A. baumannii. Macrorestriction ( SmaI) analysis of genomic DNA revealed that only one isolate was genotypically different, whereas the remaining ones were either closely related or identical. However, the genomic DNA of the A. baumannii serovar 18 reference strain proved to be genotypically unrelated.

Simplified Purification of Listeria monocytogenes Listeriolysin O and Preliminary Application in the Enzyme-linked Immunosorbent Assay (ELISA)

A simplified procedure was developed for purification of listeriolysin O (LLO) of Listeria monocytogenes, consisting of hydroxylapatite adsorption chromatography followed by Sepharose S ion exchange chromatography. The LLO (58 kDa) appeared pure in terms of sodium dodecylsulfate polyacrylamide electrophoresis and immunoblots with polyclonal rabbit immune sera. The purified LLO could be stored at -65 degrees C for 1 year without loss of immunoreactivity. Similarly, flat-bottom microtiter strips from two vendors that had been charged with LLO, could be stored at -65 degrees C for up to 3 months without loss of LLO. Three patients with documented listeriosis developed elevated IgG titres against LLO; 2 of the patients revealed minimally raised IgM titres, as determined with the enzyme-linked immunosorbent assay.

Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases.

Molecular cloning using a degenerate oligonucleotide-based polymerase chain reaction was undertaken to test the possibility that novel, developmentally regulated protein kinases are expressed in the embryonic mouse kidney. Several receptor tyrosine kinase and serine/threonine kinase cDNA clones were identified. One of these, designated DLK, represented a novel gene product whose 3.6-kilobase transcript was expressed in a tissue-specific and developmentally regulated fashion. Several clones encoding the entire open reading frame were isolated and sequenced. The identified open reading frame encodes an 888-amino acid polypeptide that defines a new subfamily within the mixed lineage protein kinase family. Sequence analysis revealed: 1) a kinase catalytic domain most characteristic of serine/threonine kinases but hybrid between members of the family of microtubule-associated protein kinase kinase kinases and the fibroblast growth factor receptor family; 2) two putative alpha-helical leucine zipper motifs separated by a 25-amino acid charged intermediate segment but lacking an NH2-terminal basic domain; and 3) COOH-terminal and NH2-terminal proline-rich domains suggestive of src homology 3 (SH3) domain binding regions. Rabbit polyclonal immune sera generated against a carboxyl-terminal bacterial fusion protein recognized a protein with an apparent molecular mass of 130 kDa in COS 7 cells that were transiently transfected with a full-length DLK cDNA expression vector. Moreover, COS 7 cells transiently transfected with an epitope-tagged DLK expression vector expressed protein with an apparent molecular mass of 130 kDa that became autophosphorylated on serine and threonine in an in vitro kinase assay.

Identification of antigens of pathogenic free-living amoebae by protein immunoblotting with rabbit immune and human sera.

Prominent antigens of pathogenic and nonpathogenic free-living amoebae were identified by using polyclonal rabbit immune sera in immunoblot assays. The intent was to determine if prominent epitopes identified with rabbit immune sera could also be recognized by human sera. With rabbit sera, the development of immunoreactive bands was restricted to molecular masses of greater than 18.5 kDa for Naegleria, Hartmannella, and Vahlkampfia antigens. Two or more broad bands of less than 18.5 kDa were prominent features in three different Acanthamoeba species. Few cross-reactive antibodies could be detected between representative species of the three different subgroups of Acanthamoeba. Naegleria antigen was likewise serologically distinct, as were Hartmannella and Vahlkampfia antigens. The relative lack of cross-reacting antibodies between the pathogenic amoebae suggested that i would be desirable to use a panel of amoebic antigens to represent the range of serologically distinct antigens for assessing reactive antibodies in human sera. In pooled human sera (10 serum specimens per pool), the appearance of minimally reactive bands ranging from 32.5 to 106 kDa was a common feature of all six antigens. A prominent band of less than 18.5 kDa was identified in the Acanthamoeba culbertsoni antigen lane in 2 of the 10 human serum specimen pools. When sera from each of the two groups were tested individually by immunoblotting, the reaction with A. culbertsoni antigen could be associated with one individual. By using a panel of amoebic antigens, this method could prove useful in recognizing undiagnosed amoebic infections by revealing specific reactive antibodies.

Purification and partial characterization of type 3 fimbriae from Salmonella typhimurium var. copenhagen

Aggregative thin fimbriae from a pigeon pathogen, Salmonella typhimurium var. copenhagen (STMVC) Mö 8 were isolated and purified. These fimbriae remained associated with the cells even after attempts to separate them from blended cells by centrifugation. After purification, fimbriae and little cell fractions were polymerized in formic acid and then analyzed by SDS-PAGE. This pretreatment resulted in the appearance of a main protein band of 17 kDa. The N-terminal amino acid sequence of 19 residues of purified 17 kDa protein showed considerable homology with the N-terminal sequence of thin fimbriae of S. enteritidis. Native fimbriae on whole cells were specifically labelled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17 kDa protein in Western blots. The polyclonal immune serum did not cross-react with the type-1 fimbriae produced by STMVC.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Characterization of HIV-1 reverse transcriptase with antibodies indicates conformational differences between the RNAse H domains of p 66 and p 15.

Antibody binding to the p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.

Reusable fiber-optic-based immunosensor for rapid detection of imazethapyr herbicide

Imazethapyr is a herbicide belonging to the imidazolinone class of compounds. It is the active ingredient of a commercial herbicide PURSUIT®. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of imidazolinone compounds. Using the immune rabbit serum, an enzyme-linked immunosorbent assay (ELISA) has been developed for the entire class of commercial imidazolinone herbicides including imazaquin, imazapyr, imazethapyr and imazamethabenz methyl. The quantitation of imidazolinones is soil requires a certain amount of sample pretreatment, thus the through-put is not ideal. A simpler immunoassay method for screening large amount of soil samples economically would be useful. Taking the sheep polyclonal immune serum, we used a fluorescent immunoassay employing optical fiber and fluorescence (US Patent 4,582,809, 1986), to assay for imazethapyr. Purified sheep antibody was immobilized on quartz fibers. A mixture of fluorescein-labelled imazethapyr analog and free imazethapyr was presented to the fiber for direct competition of the antibody binding sites or displacement of a previously fluorescein labelled fiber. The response time for the detection of imazethapyr ranged from seconds to minutes. The sensitivity of the assay was 1 nM. This binding of the fluorochrome to the fiber was reversible by washing with a phosphate buffered saline. Multiple measurements were easily processed with a single fiber over the course of several hours. Analysis of imazethapyr residue in soil can be accomplished by subjecting a clarified soil extract solution directly for analysis without further treatment. The crossreactivity data indicates that the assay is apparently specific for the imidazolinone class of compounds.