Immunobead Assay Research Articles

Ciguatoxin-like substances in edible fish on the eastern Mediterranean

Background. The consumption of edible fish (e.g., Siganus spp) was assumed to have caused ciguatera poisoning at an atypical site, the eastern Mediterranean. This pilot study assesses the presence of ciguatoxin-like substances in edible fish on the eastern Mediterranean coast of Israel. Methods. Samples of Siganus rivulatus from polluted seawater (Haifa Bay), Siganus rivulatus from relatively clean seawater (Dor), and fish from the freshwater Sea of Galilee not inhabited by toxic algae were analyzed during summertime. Ciguatoxin-like substances were tested by a membrane immunobead assay that yields a color reaction (positive, weakly positive, negative). Results. Significantly more large and small fish from Haifa Bay yielded positive color reactions compared to fish from Dor. Sea of Galilee fish gave no positive color reactions. Conclusions. Our results suggest the presence of ciguatoxin-like substances in edible fish of the eastern Mediterranean. Additional analyses are needed to determine whether these substances are ciguatoxins or related polyethers.

“Acute Phase Lipids” associated with phospholipids of Cardiolipins in sera of patients with Chronic Fatigue Syndrome (CFS) and other diseases

Interest in the study of lipids in CFS serum was initiated when it was shown that the lipids demonstrated an immunological reaction with MAb-CTX (monoclonal antibody to Ciguatoxin) associated with Ciguatera Fish Poisoning (CFP). CTX is a lipid marine toxin that is considered to be a neurotoxin. We have examined 328 CFS sera in a study with 17 CFP, 8 Gulf War, 24 Prostate Cancer, and 52 normal sera in the modified Membrane Immunobead Assay (MIA) procedure for CTX. Lipids from serum are extracted with 4 parts acetone to 1 part serum. The solution is shaken thoroughly and the suspension centrifuged at 1000 rpm for 10 minutes. The acetone phase is decanted into a tared 10 ml test tube and the acetone evaporated by a stream of air in a hood overnight. The dried lipid phase is weighed and recorded. The highest dilution showing color (+/−) with the MAb-CTX beads is scored as the end titre. A titre ≥ 1:40 is considered positive for “Acute Phase Lipids.” 328 CFS patient’s sera were examined by the modified MIA with purified MAb-CTX and 91% gave a titre ≥ 1:40.76% of the 17 CFP sera samples and 100% of the 8 Gulf War sera samples also had a titre ≥ 1:40.92% of 52 normal sera showed titres of 1:20 or less, while 4 gave titres of ≥ 1:40. In addition, 41 sera were examined for Anti-Cardiolipin (aCL) by a commercial ELISA procedure with 87.8% demonstrating IgM, IgM + IgA, or IgM +IgG aCL antibodies. These results showed mostly the IgM antibody in the samples. Preliminary chemical analyses have shown the lipids to be phospholipids associated with cardiolipin of the mitochondrial phospholipid. We designate this lipid, “Acute Phase Lipid” comparable to “Acute Phase Proteins” (CRP and SAA) in inflammatory conditions. [Supported, in part, by National CFIDS Fdn, Needham MA, and the Hokama-Yagawa Fund, UH Fdn]

Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1α, IL-1β and tumor necrosis factor (TNF)-α RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1α, IL-1β and TNF-α mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1α, IL-1β and TNF-α mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.

Biological activity of the functional epitope of ciguatoxin fragment AB on the neuroblastoma sodium channel in tissue culture

It is well established that the targeted receptor for ciguatoxin (CTX) in mammalian tissues is the sodium channel, affecting the influx of sodium into cells and altering the action potential and function of the cell. Since the syntheses of fragments of CTX has become available, our focus has been on the receptor functions of the west sphere AB and east sphere JKLM fragments using the neuroblastoma cell assay, guinea pig atrium assay, and the membrane immunobead assay (MIA). The data presented here suggest that the west sphere AB of the ciguatoxin molecule is the active portion and is responsible for the activation of the sodium channels.

Autoimmune thrombocytopenia: Determination of platelet-specific autoantibodies by flow cytometry

Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of which is one of exclusion, thus inevitably associated with potential difficulties. Current methods used to determine antigen-specific antibodies including MAIPA and the radioactive immunobead assay, are not routinely used due to methodological and practical limitations. To facilitate diagnosis, flow cytometric methods have been developed, suitable for testing a single or multiple samples. The feasible flow cytometric methods with their high sensitivity and specificity should facilitate the routine use of diagnostic methods for autoimmune thrombocytopenia and permit follow-up to determine immune remission.

Ciguatera Fish Poisoning, Canary Islands

To the Editor: Ciguatera outbreaks usually occur in the area between 35° north and 35° south latitude, mainly in the Caribbean, Indo-Pacific islands, and the Indian Ocean (1–5) (Figure). Occasionally, ciguatera poisoning has been reported outside disease endemic areas, such as the Bahamas, Canada, or Chile, but no case had been described in the West African region until now. European and Spanish cases have been rarely described and are mainly associated with seafood imported from disease-endemic regions (6). Figure Worldwide distribution of ciguatera. Gray indicates coral reef regions located between 35° north and 35° south latitudes; brown indicates disease-endemic areas of ciguatera; red circle indicates Canary Islands (latitude 28°06´ ... Ciguatera fish poisoning is a clinical syndrome caused by eating contaminated fish (1). The causative toxins of its clinical manifestations are ciguatoxins (7). These toxins are transmitted by dinoflagellates of the species Gambierdiscus toxicus, which lives adhered to damaged coral reefs in tropical seas (2). Herbivorous fish species accumulate toxins in their musculature, liver, and viscera after ingesting dinoflagellates. Larger marine carnivores eat contaminated fish and concentrate ciguatoxins (1,2). More than 425 species of fish are associated with ciguatera poisoning in humans. The most commonly implicated fish are barracuda, red snapper, grouper, amberjack, sea bass, surgeonfish, and moray (eel) (2,3). In January 2004, 2 fishermen captured a 26-kg amberjack (local name: Medregal Negro; scientific name: Seriola Rivoliana) while scuba diving along the coast of the Canary Islands, Spain. The fishermen filleted the fish and stored fillets in a household freezer. Within a few days, one of the fishermen and 4 family members consumed some fish, and neurologic and gastrointestinal symptoms developed within 30 minutes to 28 hours. The 5 family members sought treatment at the emergency room of Hospital de Fuerteventura and the Outpatient Clinic of Infectious Diseases and Tropical Medicine Service of Hospital Insular de Las Palmas. The 5 family members exhibited a combination of gastrointestinal (diarrhea [4 persons], nausea/vomiting [3 persons], metallic taste [1 person]), cardiologic (heart rhythm disturbances [2 persons]), systemic (fatigue [5 persons], itching [3 persons], dizziness (1 person]), and neurologic manifestations (myalgia [3 persons], peripheral paresthesia [3 persons], perioral numbness [2 persons], and reversal of hot and cold sensations [3 persons], which is pathognomonic of ciguatera poisoning). These clinical observations and laboratory data were collected from a prospective questionnaire filled in by physicians at the patients' first visits. No hematologic or biochemical abnormalities were detected in any patient. Based upon the symptomatic profiles, relationships of the patients, and their common dietary histories, ciguatera intoxication was diagnosed in all. None of the patients required hospitalization. The neurologic and gastrointestinal symptoms resolved over several weeks, but intermittent recurrence of some symptoms, at lower intensities, was noted for several months. A portion of the implicated fish was recovered from freezer storage at the fisherman's home. A solid-phase membrane immunobead assay with a monoclonal antibody directed against Pacific ciguatoxins and related polyether toxins was used to detect ciguatoxins or other antigenically related substances in fish tissues. Results were positive. A 150-g sample of the fish was delivered to the US Food and Drug Organization's Gulf Coast Seafood Laboratory, Dauphin Island, Alabama, USA, for sodium channel–specific in vitro assay (8) and liquid chromatography–mass spectrometry (LC/MS/MS) analysis. Assay results were positive and the ciguatoxin content of the fish sample was estimated to be 1.0 ppb (ng/g). Caribbean ciguatoxin (CCTX-1: MH+ m/z 1141.6) was confirmed by LC/MS/MS by using multiple reaction monitoring (9). The amount of ciguatoxin in the fish tissue estimated by in vitro assay was low, and close to the limit the LC/MS/MS method can detect. At least 2 additional toxins were detected in the fish sample by in vitro assay of liquid chromatography fractions. We cannot rule out the possibility that these toxins represent new ciguatoxinlike structures unique to the eastern Atlantic. Further studies are necessary to elucidate all toxins implicated in this outbreak. Classic symptoms of ciguatera developed in our patients after eating a fish they captured in the Canary Islands, which are not in the ciguatera-endemic zone (Figure). The preliminary results of this outbreak investigation suggest the presence of ciguatoxins or ciguatoxinlike structures in fish from temperate waters of the eastern Atlantic. Ciguatera poisoning is a matter of public health concern and residents of coastal West Africa and the regional island archipelagos could be a new community at risk for this seafood intoxication syndrome. We emphasize that ciguatera poisoning is a debilitating disease, and therapeutic intervention strategies are very limited (10).

TESTICULAR MICROLITHIASIS AND ANTISPERM ANTIBODIES FOLLOWING TESTICULAR BIOPSY IN BOYS WITH CRYPTORCHIDISM

Testicular biopsy (TBx) performed during orchiopexy in boys with cryptorchidism can help to predict future semen analyses and possibly identify patients at risk for testicular cancer. It has been theorized that TBx can be detrimental to the long-term health of the testis. We examined testicular microlithiasis (TM) and antisperm antibody (ASA) production in cryptorchid TBx. A total of 112 males underwent fertility evaluation. These patients had previously undergone orchiopexy and bilateral TBx (mean age 8.6 years) for unilateral or bilateral undescended testis. At a mean age of 19.6 years all patients underwent physical examination and scrotal ultrasound for evaluation of testis size, echotexture and abnormalities. Of 112 patients 57 also underwent direct Immunobead(R) assay with positive controls. Moving sperm free and/or bound to IgG bead complex were counted in the preparation. A total of 29 patients had bilateral and 83 had unilateral undescended testis. Of the 112 patients 26 were black and 86 were white. Four black patients (15.4%) and 4 white patients (4.7%) had testicular microlithiasis. Three patients had bilateral and 5 had unilateral diffuse TM. No tunica albuginea scars or testis masses were noted. In the 57 patients who underwent direct Immunobead assay no semen sample demonstrated evidence of forming bead-sperm complex, ie none had direct ASA. No patient exhibited evidence of direct ASA. Prepubertal open TBx does not increase the rate of TM. We found no evidence of additive testicular damage associated with TBx at the time of orchiopexy.

Autoimmune thrombocytopenia: flow cytometric determination of platelet-associated autoantibodies against platelet-specific receptors

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. We here describe a feasible diagnostic method using the commonly available technique of flow cytometry. An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls. The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads. The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8-31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7-1.2), and in the healthy controls 1.0 (range 0.7-1.3). The average assay coefficient of variation was 0.218 [95% confidence interval (CI) 0.213, 0.221]. The difference between the ITP patients and both groups was highly significant (P < 0.001), using a stringent non-parametric analysis. A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R2 = 0.71, 95% CI 0.39, 0.53). The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033. We conclude that the present FCM assay is clinically useful for routine diagnosis and follow-up of ITP.

Prospective evaluation of the immunobead assay for the diagnosis of adult chronic immune thrombocytopenic purpura (ITP)

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-induced platelet destruction. To better define the role of antigen-specific assays in adult chronic ITP, we prospectively measured platelet-associated autoantibody against either glycoprotein (GP) IIb/IIIa or GPIb/IX in 282 patients with chronic ITP and 289 patients with thrombocytopenia of other causes. We divided chronic ITP into four subgroups: presplenectomy, mild (platelet count >30 000 micro (L-1) requiring no therapy), presplenectomy, severe (platelet count <30 000 micro L(-1) requiring therapy but not splenectomy), postsplenectomy, remission (postsplenectomy partial or complete remission without further therapy) and postsplenectomy refractory (required therapy after splenectomy failure). Positive results: total ITP group, 55.4%; presplenectomy, mild, 31.1%; presplenectomy, severe, 42.6%; postsplenectomy, remission, 50.0%; and postsplenectomy, refractory, 87.8%. In addition, the degree of positivity increased with the severity of the patient's disease. The assay had a minimum specificity of 84.4% if clinical factors, consistent with immune thrombocytopenia, were not considered in patients with thrombocytopenia associated with other diseases. However, if clinical factors consistent with immune thrombocytopenia were considered and only patients with questionable immune thrombocytopenia and patients 'lost to follow-up' were included in the false-positive group the specificity was 93.1%. We conclude that the presence of immune thrombocytopenia is highly probable if the immunobead assay is positive and that antigen-specific assays are diagnostically useful in adult chronic ITP.

Chronic phase lipids in sera of chronic fatigue syndrome (CFS), chronic ciguatera fish poisoning (CCFP), hepatitis B, and cancer with antigenic epitope resembling ciguatoxin, as assessed with MAb-CTX.

Clinical reports and descriptions of chronic fatigue syndrome (CFS) and chronic ciguatera fish poisoning (CCFP) show great similarities in clinical symptomology. These similarities in the literature suggested the exploration of lipids in sera of CFS, CCFP, and other diseases with the membrane immunobead assay (MIA), which is typically used for screening ciguateric ocean fish. Sera from patients with other diseases, including hepatitis B, cancer, and diabetes, were included to assess the degree of specificity involved. Sera were treated with acetone in a ratio of 1 part serum to 4 parts acetone. The suspension was centrifuged, and the acetone layer was evaporated. The residue was weighed and redissolved in 1.0 mL methanol and tested by the MIA, undiluted and titered to 1:160. The undiluted acetone fraction of the 37 normal showed +/- activity to +activity with 16 no titer, 15 with 1:5 titer and two with 1:10 titer, and four with > or =1:40 titers. One hundred fifteen CFS sera showed 1 with 1+ and 114 with 2+ activity in the undiluted samples, 1 with 1:10 titer, 3 with 1:20 titer, 31 with 1:40 titer, 50 with 1:80 titer, and 30 with 160 titer. Thus 95.6% of the samples had > or =1:40 titer. Eight hepatitis B sera samples had > or =1:40 titers. Four CCFP samples had > or =1:40 titers. Three of 16 cancer samples had 1:40 titer. These data are summarized in Fig. 1. As shown in Table 1, a significant increase (P<0.001) in the chronic phase lipids (CPLs) was shown relative to the normal group. A preliminary chemical study in C18 octadecylsilyl columns showed all fractions (100% chloroform, 9:1 chloroform : methanol, 1:1 chloroform : methanol, and 100% methanol) to contain lipids reactive to MAb-CTX with different intensities. Prostaglandins were shown in 100% methanol fraction. Competitive MIA with crude fish ciguatoxin and CFS with synthetic JKLM ciguatoxin epitope suggested similarities in structure with ciguatoxin. This was compatible with the neuroblastoma assay demonstrated in the C(18) column fractions 9:1 and 1:1, chloroform : methanol solvents.

Chronic Phase Lipids in Sera of Several Chronic Diseases Reacting with MAB–CTX (Antibody to Ciguatoxin)

The membrane immunobead assay results on the acetone lipid fraction of serum from chronic fatigue syndrome (CFS) patients (60 samples) and normal individuals (with no clinical CFS or other disease symptoms) showed significant differences with 4 exceptions (4 normals showed 1:40 and 1:80 titres). This represented approximately 10.8% of the normal samples, with 3 samples at 1:20, the majority of the CFS titred 1:40 through 1:160. This represented 95.0% of the samples. The small numbers of hepatitis patients and chronic ciguatera fish poisoning patients also had titres of 1:40 to 1:80 in all of the serum samples examined. The weights of the lipids in mg/ml serum essentially are very similar, except 1 or 2 of CFS and hepatitis B showed values at the upper level. Comparison of sexes showed 65% females and 35% men with CFS, representing a ratio of approximately 2:1 (female/male). It is concluded that certain disease conditions and environmental exposures to deleterious factors (toxin, chemicals, microorganisms)...

Immunomagnetic DNA Aptamer Assay

DNA aptamers, oligonucleotides with antibody-like binding properties, are easy to manufacture and modify. As a class of molecules, they represent the biggest revolution to immunodiagnostics since the discovery of monoclonal antibodies. To demonstrate that DNA aptamers are versatile reagents for use as in vitro diagnostic tools, we developed a hybrid immunobead assay based on a 5'-biotinylated DNA thrombin aptamer (5'-GGTTGGTGTGGTTGG-3') and an anti-thrombin antibody (EST-7). Our results show that the thrombin DNA aptamer is capable of binding to its target molecule under stringent in vitro assay conditions and at physiological concentrations. These findings also support the view that DNA aptamers have potential value as complementary reagents in diagnostic assays.

Fertilization antigen-1 removes antisperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction

Objective: To determine if fertilization antigen (FA)-1 will remove autoantibodies from the surface of sperm cells of immunoinfertile men by immune adsorption and permit an increased acrosome reaction (AR). Design: Prospective analytic study. Setting: University medical center. Patient(s): Men from 18 infertile couples with autoantibodies present on their spermatozoa. Intervention(s): Sperm samples after processing were examined for antibody binding and AR before and after adsorption with control medium or FA-1. Main Outcome Measure(s): Sperm-bound antibody was assessed by the immunobead assay (immunoglobulin [Ig] A and IgG) and the AR by induction with ionophore A23187. Result(s): Adsorption with FA-1 compared with control medium increased immunobead-free swimming sperm an average of 50% and 76% for IgA and IgG antisperm antibodies, respectively, with 78% and 100% of the 18 semen specimens increasing significantly. The AR rate increased an average of 10.3% compared with control medium and showed improvement in 78% of the sperm samples after FA-1 adsorption. Conclusion(s): The FA-1 sperm antigen appears to significantly free sperm cells coated with autoantibodies in the semen of most infertile men examined. Reducing sperm-bound antibodies that inhibited the AR allowed the sperm cells to undergo successful AR induction by calcium ionophore.

A Simple Membrane Immunobead Assay for Detecting Ciguatoxin and Related Polyethers from Human Ciguatera Intoxication and Natural Reef Fishes

A simple membrane immunobead assay (MIA) for detecting ciguatoxin (CTX) and related polyethers directly from fish tissue is presented. A membrane laminated onto a solid plastic support is immersed with a piece of fish tissue in methanol. The membrane is thoroughly dried and placed into an immunobead suspension containing polystyrene particles coated with monoclonal antibody to CTX (MAb-CTX). Two beads of different diameter and color are used. The color intensity of the membrane is related to the concentration of the toxin bound to the membrane. Twelve of 13 fish implicated in human ciguatera fish poisoning showed borderline or positive responses in the assay. A Sphyraena barracuda sample that tested negative with the MIA and was highly toxic with the mouse toxicity bioassay showed only weak CTX-like toxin activity in the guinea pig atrial assay, indicating that the major toxin in the sample was not CTX-like. Examination of 154 routinely caught reef fish from Hawaii, Kosrae, and Kwajalein by MIA found 132 (86%) negative and 8 (5%) positive for CTX, with 14 (9%) giving a borderline response. Fish from Hawaii showed a higher frequency of borderline or positive responses than those from Kosrae and Kwajalein, probably because several species of fish from several islands of Hawaii were tested, whereas only one species from a single area was examined from each of the islands of Kosrae and Kwajalein.

PARTIAL OBSTRUCTION, NOT ANTISPERM ANTIBODIES, CAUSING INFERTILITY AFTER VASOVASOSTOMY

Purpose We determined whether men who may have partial obstruction and antisperm antibodies after vasovasostomy can be distinguished from other infertile men with antisperm antibodies only, and whether repeat microsurgical reversal is beneficial in such patients. Materials and Methods A total of 412 patients underwent indirect immunobead testing for antisperm antibodies at our laboratory from December 1991 through July 1996. Of 95 patients with an assay greater than 20% binding 49 had normal partners and were grouped by history of vasovasostomy (20), varicocele (9), cryptorchidism (8) and epididymo-orchitis (12). Semen analysis characteristics and antisperm antibody binding variables were compared across histories. Pregnancy rates were compared between patients treated surgically for partial obstruction and those treated for antisperm antibodies. Mean followup was 33.8 months. Results Compared to the other 3 groups, men with a history of vasectomy and reversal had significantly lower sperm concentration (p = 0.002), poorer motility (p <0.001), lower overall binding on the indirect immunobead assay (p <0.001) and lower IgA binding (p = 0.008). The clinical diagnosis of partial obstruction was based on a sense of epididymal fullness by palpation, as well as the aforementioned semen parameters. Of the 20 patients with a history of vasectomy and reversal 14 were diagnosed with partial obstruction and underwent repeat microsurgical reversal and 6 with a history of vasovasostomy but no evidence of obstruction received no further therapy and never established pregnancies. The remaining 29 patients underwent sperm washing and assisted reproduction. Of 14 patients 7 (50%) established pregnancies after repeat reversal compared to only 5 of 29 patients (17.2%) treated with assisted reproduction (P = 0.025). Conclusions Antisperm antibodies are not a significant factor in persistently infertile post-reversal cases with the aforementioned criteria. Repeat reversal appears to be the most successful treatment option in this setting.

Quantitation of platelet-specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens.

Immune thrombocytopenic purpura (ITP) is a disorder caused by anti-platelet autoantibodies (Ab), most of which are directed against epitopes on platelet membrane glycoprotein complexes GPIIb/IIIa and GPIb/IX. To detect platelet Ab, reliable techniques, such as MAIPA or immunobead assay, have been developed. They all achieve their selective specificity by the use of monoclonal antibodies (MoAb) against defined glycoproteins of the platelet membrane. In order to determine the most frequent Ab-specificities, a novel enzyme-linked immunosorbent assay, named platelet-glycoprotein-ELISA (P-GP-ELISA), has been developed. It uses purified GPIIb/IIIa and GPIb/IX complexes, respectively, as antigens and enables determination of platelet-associated as well as circulating Ab (IgG, IgM). MoAbs are not required and therefore there is no risk of competition between MoAb and Ab. Levels of Ab in patients with the clinical diagnosis of an idiopathic thrombocytopenic purpura were analysed. 92.7% (76/82) platelet eluates with significantly increased levels of Ab against at least one of the glycoproteins were found, whereas no sample from healthy volunteers (0/37) gave a positive result, pointing to a high sensitivity and specificity of the test system. Since its application is also easy and quick, P-GP-ELISA should facilitate detection of Ab against platelet membrane proteins in routine determinations.

Immunosuppressive activities in the seminal plasma of infertile men: relationship to sperm antibodies and autoimmunity.

Semen samples from infertile men were assessed for sperm autoimmunity by direct immunobead assay for immunoglobulin (Ig)A and IgG sperm antibodies and mucus penetration test. Immunosuppressive activity in seminal plasma was measured by an in-vitro bioassay employing dose-dependent inhibition of phytohaemagglutinin-induced activation of rat thymocytes, in the presence or absence of hydroxylamine (0.1 mM), an inhibitor of polyamine oxidation. All seminal plasma samples, regardless of autoimmune status, caused inhibition of T-lymphocyte activation, and hydroxylamine reduced this bioactivity by appproximately 50%. Dialysis (<3500 molecular weight) also significantly reduced seminal plasma bioactivity, both in the presence and absence of hydroxylamine. In the presence of hydroxylamine, there was a negative correlation between IgA, but not IgG, antibody concentrations and lymphosuppressive activity in seminal plasma. Antibody-positive samples displaying impaired sperm function, as indicated by the mucus penetration test, had reduced activity compared with other samples. In contrast, there was no relationship between sperm autoimmunity and lymphosuppressive activity assayed in the absence of hydroxylamine. The data indicate that T-lymphocyte inhibition by human seminal plasma is due to multiple factors, and reduced amounts of these factors may contribute to the development and/or persistence of sperm autoimmunity in infertile men; however, differences in polyamine substrates available for oxidation in semen do not appear to be a major contributing factor.

Application of immunomagnetic beads in combination with RT-PCR for the detection of circulating prostate cancer cells.

Recently published protocols using Reverse Transcriptase Polymerase Chain reaction (RT-PCR) for prostate specific antigen (PSA) provide a sensitive means for detecting circulating prostate cancer cells. Attempts to use these assays for staging of prostate cancer have produced conflicting results. As a first step towards rectifying these discrepancies, a modified immunobead-RT-PCR assay capable of detecting as few as 10 prostate cancer cells in 8cc of blood was developed. This 10 fold increase in sensitivity was achieved in part by introducing two target cell enrichment steps. As a model system to assess sensitivity of the modified assay, template RNA was extracted from PSA positive human carcinoma cells suspended in human blood and isolated with immunomagnetic beads following incubation with an epithelium specific antibody. After 45 cycles of PCR, product from as few as 10 target cells could be readily detected when displayed on a 2% agarose gel stained with SYBR Green fluorescent dye. The identity of amplified DNA fragments was confirmed by Southern blot hybridization. When applied to blood samples from patients with proven metastatic disease, the immuno-bead RT-PCR assay was successful in detecting circulating PSA positive epithelial cells, suggesting this assay may be useful for assessment of disease progression or recurrence.

Detection of Ciguatera and Diarrheic Shellfish Toxins in Finfish and Shellfish With Ciguatect Kit

Abstract The Ciguatect test kit for the detection of ciguatoxin, okadaic acid, and related polyether compounds, based on solid-phase immunobead assay technology, was developed for rapid screening of toxic fish and shellfish in harvesting areas and the marketplace. Earlier formats, based on radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) technology, were used successfully to identify and separate ciguatoxic fish harvested from Hawaiian waters. The new format utilizes a membrane attached to a plastic strip, immunobead solution, and a user-friendly readout. Multiple formats of the test kit, including a rapid extraction method (REM) and ELISA, were demonstrated to be more sensitive than the mouse assay for detection of ciguatera-related compounds. Depending on the format used, the level of detection can be as low as 50 pg okadaic acid per gram of tissue. The test kit has been used to map ciguatoxic and nontoxic fishing areas around the islands of Hawaii through the use of a biomarker and the monitoring of toxic fish obtained from Pacific and Caribbean regions. Ciguatera-related toxins can be detected directly on fish flesh or after extraction by traditional procedures or the newly developed REM. Initial applications of the test kit to monitor diarrheic shellfish poisoning (DSP)-related toxins in mussels before, during, and after Dinophysis sp. blooms and during depuration operations were successful. The Ciguatect methodology can be used to monitor reef fishing areas for ciguatera potential, shellfish beds for DSP toxins, and shellfish depuration operations for elimination of DSP toxins and to screen for toxic fish and shellfish in the marketplace.

Variation in Antisperm Antibody Results Using Different Assays

The detection of various types of antisperm antibodies (ASA) in the serum varies among different assays. This variation may influence the diagnosis and management of infertile couples who are tested for such immunologic factors. This prospective study was conducted to determine the variation in the results of ASA as measured by the sperm immobilization (SI), sperm agglutination (SA), and the indirect immunobead (IB) assays. The sera of 79 patients that tested positive for ASA by at least one of the assays listed above were concurrently tested with all three assays. Using an individual ASA assay, 66 (84%), 26 (33%) or 36 (46%) of sera tested positive by the SA, SI, or IB assays, respectively. However, using a combination of assays, 67 (85%), 78 (99%) or 40 (51%) of sera tested positive using either the SA+SI, SA+IB or SI+IB assays, respectively. These findings suggest that the utilization of different assays to detect ASA may detect sera that are positive for ASA with more reliability than single assay testing.