Enhanced specificity for small molecules in a convenient format which removes a limitation of competitive immunoassay.

Anti-complex immunoassay systems for small molecules permit the exquisite specificity achievable with monoclonal antibodies to be expressed to an extent which is not possible with competitive format immunoassay. While our previously reported anti-complex system is superior to competitive systems in terms of sensitivity, precision and specificity we have found that this specificity may be enhanced dramatically by simply interposing a wash step between the addition of sample and that of the labelled anti-complex antibody. When such a wash step was attempted with the competitive format system, after addition of sample but before addition of the labelled component, assay performance was degraded to the extent of making it unusable. We suggest, therefore, that the inherent flexibility of the anti-complex approach for small molecule assay creates an opportunity for remarkable enhancement of the functional specificity of primary antibody.

Ultra-specific immunoassays for small molecules: roles of wash steps and multiple binding formats.

New immunometric forms of immunoassay are much more flexible to use than competitive-format immunoassays for small molecular analytes. An example of the utility of this flexibility is the ability to wash the capture antibody after it has been exposed to analyte but before addition of the labeled reagent. This simple maneuver has a large impact on the specificity obtained from already highly specific assays. We also show that specificity can be further increased by means of our multiple binding assay approach, in which the final reading reflects analyte binding to two different primary capture monoclonal antibodies.

High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin.

We report the first clinical application of a noncompetitive immunometric assay system that provides advantages for the rapid and robust assay of small molecules similar to those realized for larger molecules with two-site immunometric assays. This anti-immune complex assay is based on the interaction of a receptor such as a primary antibody with its ligand, such that new binding sites, recognizable by a secondary antibody, are formed. In this report the system is applied to the measurement of digoxin in serum. Utilizing an anti-complex antibody that recognizes a digoxin-bound primary antibody with affinity > 2000-fold over its binding to the primary antibody alone, we show that this anti-complex assay system provides a high-performance assay for serum samples, being conveniently simple (immobilized primary antibody binds digoxin and then labeled secondary antibody so that when excess unbound label is washed away the immunometric readout reflects the digoxin concentration), rapid (incubation time 1-10 min), sensitive (detection limit 30 ng/L), precise (3-4% within-run CV, 1-8% total CV), and free from interference from digoxin-like immunoreactive factors.

Kinetics of expression of two major Plasmodium berghei antigens in the mosquito vector, Anopheles stephensi.

Expression of a 21 kDa determinant (Pbs21), first detected on the surface of ookinetes, and of the circumsporozoite protein (CSP) was studied by immunofluorescence and Western blots during the developmental cycle of Plasmodium berghei in the mosquito Anopheles stephensi. The expression of Pbs21 was predominantly localised on the ookinete surface one day after the infectious blood meal, and thereafter reactivity declined to a minimum on days 2 and 3, the time of onset of oocyst development. A gradual increase in fluorescence was observed on the oocysts from day 6 that was retained until day 17 post-infection. In contrast, sporozoites released from oocysts or salivary glands showed little or no antibody labelling with anti-Pbs21. Circumsporozoite protein was not detectable in any midgut preparations until 5-6 days after feeding, when reactivity was observed against immature oocysts. Expression then continued and increased throughout oocyst and sporozoite development. Western blots confirmed that Pbs21 was expressed minimally during the oocyst development but was not detectable in sporozoites. Co-localisation of anti-Pbs21 and anti-CSP monoclonal antibodies to the 50 kDa and 60 kDa bands in Western blots of sporozoite suggests immunological cross-reactivity between the CSP and the anti-21 kDa antibodies.

Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.

Immunoprotection in mice susceptible to waning memory against the pre-erythrocytic stages of malaria after validated immunisation with irradiated sporozoites of Plasmodium berghei.

The induction of immunity by irradiated sporozoites has been a bench-mark of immunological protection against the malaria parasite. Herein we confirm that different mouse strains exhibit different susceptibilities to sporozoite-induced infection of Plasmodium berghei. We note, however, that after hepatic schizogony, early parasite growth in the blood demonstrates no strain preference between C57BL/6 and BALB/c mice. Sporozoite-susceptible C57BL/6 mice, although initially protected by irradiated sporozoite immunisation against a challenge of 10(3) live sporozoites, progressively lose this protection; a challenge with fewer sporozoites 2 months later elicits a blood infection. BALB/c mice treated in parallel remain protected. Analysis of the kinetics of blood parasitaemia (a measure of hepatic schizont burden) with waning protection shows clearly that immunocompetence remains, as indicated by a reduction in the effective exo-erythrocytic schizont load. This immunocompetence can be shown to be absolutely protective, given an appropriately low dose of viable infective sporozoites. We discuss the testable proposition that this elicitation of protective memory is a consequence either of 'unsaturated' threshold levels of recirculating immunoeffector CD8+ cells or of CD4 cell activation by nonviable sporozoites.

The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy.

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

Detection of mature malaria infections in live mosquitoes.

A method has been developed which detects malaria parasites in the salivary glands of live Anopheles stephensi. The method exploits the sugar feeding behaviour of the mosquito and requires only routine Western blotting techniques on nitrocellulose membrane (NCM). Infectivity can be determined without any direct manipulation of individual mosquitoes. Female A. stephensi were infected with the rodent malaria parasite, Plasmodium berghei, and after 14-16 d were starved of fructose overnight (12-18 h), then resupplied with fructose presented through a small piece of NCM. Mosquitoes were allowed to probe the membrane for several hours; the NCM was then removed and subjected to a standard immunoblotting protocol using an anti-P. berghei circumsporozoite protein (CSP) monoclonal antibody as the primary reagent, and a horseradish peroxidase-coupled secondary antibody. NCMs taken from cages containing infected mosquitoes showed a variable number of small black dots where individual females had probed and deposited either CSP or sporozoites. Infectivity could be detected easily from 13-14 d after feeding, and in as few as 10 mosquitoes at 19 d after infection; in one instance, infection in a single mosquito was clearly determined. After blocking with goat serum, the NCMs could be stored for 3-4 months and still provided positive reactions, offering some potential for applicability to field research studies.

Analysis of immunity induced by the affinity-purified 21-kilodalton zygote-ookinete surface antigen of Plasmodium berghei.

By using affinity-purified ookinete surface antigen from the rodent malaria parasite Plasmodium berghei, a transmission-blocking immunity was induced in mice. Groups of mice were immunized via different routes, with total quantities of antigen ranging from 0.5 to 40 micrograms (with or without Freund adjuvant). Vaccination by the intramuscular route with 20 micrograms of antigen in the absence of adjuvant and boosted once with the same amount of protein induced a total transmission blockade. Immunoblot analysis confirmed that immune sera invariably recognized Pbs21 antigen. The isotype and titer of the anti-Pbs21 immunoglobulin G (IgG) response was determined by enzyme-linked immunosorbent assay. The antibody isotype was predominantly IgG1. The concentration of specific anti-Pbs21 IgG reached a peak of 182.45 +/- 92.13 micrograms/ml by week 7 postimmunization and fell progressively to 38 micrograms/ml at week 34 (at which time the transmission was still inhibited by 98%).

An antigen specific to the liver stage of rodent malaria recognized by a monoclonal antibody.

Vaccines currently being evaluated against malaria are based on proteins derived from the blood, sporozoite and sexual stages. Antigens from the liver stage, which is now recognized as the major target of protective sporozoite induced immunity, have received comparatively little attention. This paper describes the generation of a monoclonal antibody (MoAb), which recognizes an antigen specific to the liver stage of the rodent malaria Plasmodium berghei. The antigen is expressed throughout liver stage development and appears to be localized to the parasitophorous vacuole membrane. The MoAb did not affect the growth of liver stages cultured in vitro nor could protection be demonstrated in vivo following passive transfer of the antibody.

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.

A liver-stage specific antigen of P. berghei identified by a monoclonal antibody.

Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each stage are being tested for their potential as vaccine candidates. Recently it has become clear that the liver stage can also be the target of protective immune responses; however, only the circumsporozoite protein has been identified as a protective liver antigen. It is critical for vaccine evaluation and development to identify other liver antigens and assess their potential role in immunity. In this paper we describe a monoclonal antibody, which recognizes a liver-specific antigen of Plasmodium berghei (referred to as Pbl1). Passive immunization studies using this antibody suggest that it may influence the course of sporozoite-induced infections.

Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of rodent malaria.

The time and site of expression of five antigens, recognized by monoclonal antibodies raised against blood-stage parasites, were studied in the exoerythrocytic stage of Plasmodium berghei using indirect immunofluorescent antibody staining. Two monoclonal antibodies (W 3.5, I 2.6), which stain the cytoplasm of infected erythrocytes, did not stain the cytoplasm of the infected liver cell but stained the parasite itself suggesting a difference in the antigenic architecture of the erythrocytic and exoerythrocytic parasites. Another antibody (17.6.1) revealed a further difference in the antigenic composition of the blood and liver-stage parasites being expressed almost exclusively in the former. Two others (C139 and 17.3.9) showed broadly similar patterns of expression in these two stages of the malarial life-cycle.

Improved techniques for the culture of the liver stages of Plasmodium berghei and their relevance to the study of causal prophylactic drugs.

The in vitro exoerythrocytic (EE) of Plasmodium berghei was compared in primary rat and mouse hepatocytes and the human hepatoma cell line HepG2. All of the cell-types supported the full maturation of EE stages, but the HepG2 cells were much more susceptible to infection than the primary rodent hepatocytes and were also the most efficient host cells. Following refinement of culture techniques, the development of EE forms which is now observed in HepG2 cells closely reflects that occurring in vivo with respect to the morphology and size of parasites and their rate of maturation. Furthermore, high densities of parasites are reproducibly achieved. A detailed description is presented of exoerythrocytic development in HepG2 cells. The application of these cultures to chemosensitivity studies is discussed and the relative advantages of employing cell lines or primary hepatocytes as host cells in such a system are considered.

Ookinete antigens of Plasmodium berghei. Appearance on the zygote surface of an Mr 21 kD determinant identified by transmission-blocking monoclonal antibodies.

Zygotes and ookinetes of the rodent malaria Plasmodium berghei can be enriched 50-fold, from whole blood cultures by ammonium chloride lysis. Three monoclonal antibodies (MoAbs) raised against such enriched preparations specifically bind to a determinant of Mr 21 kD as assessed by 125I-labelled goat anti-mouse IgG probed immunoblots of Western transfers of SDS-PAGE gels. Indirect immunofluorescence indicates that the 21 kD determinant bound by specific MoAbs, whilst not detectable on gametocytes or gametes, appears on the parasite surface within 2 h of exflagellation/fertilization and increases thereafter. The three MoAbs specifically binding the 21 kD determinant block oocyst development in mosquitoes by at least 90%, as assessed either by in-vitro membrane feeds or by live feeds on passively immunized mice. These MoAbs reduce ookinete formation in vitro by between 52 and 100%. Possible mechanisms of action of these MoAbs are discussed.

Ookinete antigens of Plasmodium berghei: a light and electron-microscope immunogold study of expression of the 21 kDa determinant recognized by a transmission-blocking antibody.

The expression of a 21 kDa transmission-blocking determinant on the malarial parasite Plasmodium berghei was studied by using the immunogold method at the light, scanning-electron and transmission-electron microscope levels. The determinant was shown to be expressed exclusively on the macrogamete and its immediate progeny the zygote, ookinete and oocyst. It is first detected on the plasmalemma two hours after the escape of the parasite from the red blood cell, reaches a maximal density on the young ookinete some ten hours later, and is still found on the oocyst after six days. The antigen is distributed evenly over the entire surface of the zygote and ookinete, but is readily shed from the parasite surface. The general applicability of the silver-enhanced immunogold method in parasitological research is emphasized.

The selection and characterization of human monoclonal antibodies to human cytomegalovirus.

This communication describes the application of Epstein-Barr virus lymphocyte transformation technology to the production of human monoclonal antibodies specific for human cytomegalovirus. A group of such IgG antibodies have been characterized in terms of subclass, light-chain composition, specificity for particular viral proteins and neutralizing capacity. These results have shown that the production of antibodies by transformed lymphocytes is representative of the in vivo human immune response; the antibodies produced may therefore be of therapeutic value. This approach should prove to be useful for the identification of specific virion proteins which are antigenic in humans and for the in vitro evaluation of the immune responses to synthetic peptide vaccines.

The development of Plasmodium ookinetes in vitro: an ultrastructural study including a description of meiotic division.

Ookinetes have been cultured in vitro using modifications to the method of Weiss & Vanderberg (1977). Significant improvements in technique were produced by culture in medium at pH 8.4 and at a blood dilution at or over 1/10. Ookinetes produced were infective to mosquitoes by membrane feeding techniques. Ultrastructural analyses were made of nuclear, cytoskeletal, crystalloid and microneme development. The first intranuclear division in the zygote has been recognized as meiosis. Chromosome condensation during prophase follows the classical stages of leptotene, zygotene and pachytene. Diplotene and diakinesis are not present - the synaptonemal complexes persist into metaphase I. Chromosomes separate at anaphase and rapidly de-condense prior to telophase. We have not recognized a second meiotic division in the ookinete. The implication of these findings to the molecular and Mendelian organization of the parasite genome are discussed.