A simple radioactive binding assay for the detection of rheumatoid factor in serum

A simple radioactive binding assay for the detection of rheumatoid factor (RF-RBA) is described. Test sera are complement inactivated by incubation in 0.13 M ethylene diaminetetraacetic acid (EDTA) at 37°C and then incubated with ¹²⁵I-labelled heat-aggregated with 2.5% (w/v) polyethylene glycol 6000. Sera from 78 patients and 24 controls were tested in the RF-RBA assay and the results compared with those obtained by the rheumatoid latex test and the rheumaton test. 37 sera were positive and 59 sera were negative for rheumatoid factor by the 3 methods used. A positive correlation (r = 0.56, P < 0.01) was observed between the rheumaton titre and the RF-RIA result.     

Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis

Objectives

To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections.

Primary interaction between antibody and components of Alternaria: II. Antibodies in sera from normal,allergic, and immunoglobulin-deficient children

Antibodies in sera from normal, allergic, and immunoglobulin-deficient children were studied for binding to radiolabeled components of Alternaria tenuis. Significant binding levels were found in 103 of 105 sera from normal children. The levels were age-dependent, rising from a low point in the 7- to 12-month age group to adult levels by the age of 8 years. Levels of binding to two antigens, a culture filtrate derivative (¹²⁵I-CLF) and a mycelial derivative (¹²⁵I-IIS), were similar. Sera from asthmatic children with strong immediate skin test reactions to Alternaria extracts bound significantly higher levels of ¹²⁵I-CLF than did sera from allergic children with negative skin tests or from control children. Binding levels in sera from children with hypogammaglobulinemia were significantly less than binding levels in sera from normal children in any age group. Sera from children with selective IgA deficiency bound ¹²⁵I-CLF at normal levels. The almost universal occurrence of anti-Alternaria antibodies in children was partly explained by the finding of partial cross-reactivity and/or shared antigens among several fungal species, including A. tenuis, Stemphylium sp., Curvularia sp., and Aspergillus fumigatus. The biological significance of these antibodies is not clear, but the procedures described lend themselves to further investigations.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.     

Western Immunoblotting for Bartonella Endocarditis

To differentiate infectious endocarditis (IE) from other Bartonella infections and to identify infecting Bartonella bacteria at the species level on a serological basis, we used Western immunoblotting to test sera from 51 patients with Bartonella IE (of which 27 had previously benefited from species identification by molecular techniques), 11 patients with chronic Bartonella quintana bacteremia, and 10 patients with cat scratch disease. Patients with IE were Western blot positive in 49 of 51 cases, and significant cross-reactivity with three heterologous Bartonella antigens was found in 45 of 49 cases. Sera from bacteremic patients did not react with more than one heterologous antigen, and sera from patients with cat scratch disease gave negative results. Sera reacted only with B. henselae in four cases of IE, including one with a positive PCR result for valve tissue. Western blot and cross-adsorption performed on serum samples from patients with IE (the identity of the causative species having been determined by PCR) were demonstrated to identify efficiently the causative species in all cases. When applied to patients diagnosed on the basis of serological tests only, this technique allowed identification of the causative species in 20 of 22 cases. The results were in accordance with epidemiological features. Six reactive bands of B. quintana (of molecular sizes from 10 to 83 kDa) demonstrated significant association with sera from patients with B. quintana endocarditis. Overall, Western blotting and cross-adsorption made it possible to identify the causative species in 49 of 51 (96%) IE cases.     

Anti-Nocardia brasiliensis antibodies in patients with actinomycetoma and their clinical usefulness

Anti-Nocardia brasiliensis antibodies quantification and its clinical utility was confirmed in this study. A protein cellular extract from a N. brasiliensis strain named HUJEG-1 and registered at the ATCC # 700358 was used in a western blot assay to identify the immunodominant antigens. The protein P24 was selected to set up an ELISA test because it exhibit no cross-reaction with sera from tuberculosis and leprosy patients. A purified protease was also used as antigen in the ELISA test to compare its utility. Sera from N. brasiliensis mycetoma persons gave absorbance values above 0.3 when the disease was active using the P24 as antigen, these values decreased after patients completed their medical treatment. Anti-protease antibodies showed great variation and absorbance values similar to the healthy controls. We confirmed the clinical usefulness of the ELISA test both in serodiagnosis and in assessing the response to medical treatment. This is the first sensitive and specific serologic test for routine clinical laboratory.     

Binding of 125I myelin basic protein by serum and cerebrospinal fluid

A sensitive gel filtration redioimmunoassay was used to test for antibodies to the basic protein of myelin, the antigen of experimental autoimmune encephalomyelitis, in serum and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and other diseases. Sera and CSF from patients with MS gave results similar to those for controls including subjects with various neurological diseases. Binding of ¹²⁵I-basic protein by seven-fold concentrated CSF was shown to be due to α-globulin. Free basic protein, as a possible auto-immunogen, was sought in the serum and seven-fold concentrated CSF of patients with MS and controls by competitive inhibition in the radioimmunoassay, but none was demonstrable.

If an immunological mechanism is to be invoked in the initiation of destruction of the basic protein of myelin in MS, then either pathogenic `antibody' must be absorbed in vivo to the target antigen in the central nervous system or the initiating events must be mediated entirely by sensitized lymphocytes.

     

Detection of the 70-Kilodalton Histoplasma capsulatum Antigen in Serum of Histoplasmosis Patients: Correlation between Antigenemia and Therapy during Follow-Up

Histoplasmosis is an important systemic fungal infection, particularly among immunocompromised individuals, who may develop a progressive disseminated form which is often fatal if it is untreated. In such patients, the detection of antibody responses for both diagnosis and follow-up may be of limited use, whereas the detection of Histoplasma capsulatum var. capsulatum antigens may provide a more practical approach. We have recently described an inhibition enzyme-linked immunosorbent assay (ELISA) for the detection in patients’ sera of a 69- to 70-kDa H. capsulatum var. capsulatum-specific antigen which appears to be useful in diagnosis. To investigate its potential for the follow-up of histoplasmosis patients during treatment, antigen titers in the sera of 16 patients presenting with different clinical forms of histoplasmosis were monitored at regular intervals for up to 80 weeks. Sera from four of five patients with the acute form of the disease showed rapid falls in antigenemia, becoming antigen negative by week 14 (range, weeks 10 to 16). Sera from four patients with disseminated histoplasmosis showed falls in antigen levels; three of them became antigen negative by week 32; the fourth patient became negative by week 48. In contrast, antigen titers in four of six AIDS patients with the disseminated form of the disease remained positive throughout follow-up. Sera from only one patient who presented with the chronic form of the disease were analyzed, and this individual’s serum became antigen negative by week 9. The inhibition ELISA is shown to be of particular use in the monitoring of non-AIDS patients with the acute and disseminated forms of the disease and may complement existing means of follow-up.     

Brucella abortus RB51 and Hot Saline Extract from Brucella ovis as Antigens in a Complement Fixation Test Used To Detect Sheep Vaccinated with Brucella abortus RB51

The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 × 10⁹ CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect B. ovis-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with B. ovis also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type Brucella standard antigens.     

Evaluation of three serological tests for detection of antibody toPseudomonas aeruginosa in human sera

Four hundred and ninety-five sera from 325 patients from whomPseudomonas aeruginosa had been isolated and 86 control sera were tested for antibody by indirect haemagglutination tests (HAT) and complement fixation tests (CFT) using a polyvalent pseudomonas serotype-specific vaccine antigen, PEV-02. Sera were also tested by countercurrent immunoelectrophoresis (CIE) for precipitins to a species-specific protein antigen. Control sera gave titres of 160 or less by HAT and 20 or less by CFT. 2-Mercaptoethanol resistant antibody titres (immunoglobulin G) were below 40 for all control sera and none of the latter contained precipitins to common antigen. Of 325 patients, 156 (48%) gave titres of 320 or greater by HAT and of these, 114 (73%) showed elevated immunoglobulin G titres. Less patients with positive blood cutures than expected were positive by HAT and more patients with bone infections gave raised immunoglobulin G titres than expected. Cystic fibrosis patients were invariably seropositive by all tests. There was a correlation between positive CIE and CFT tests, especially in patients who were positive by HAT. Approximately half of 83 patients tested gave a serotype-specific antibody response. The tests were of little value in confirming clinically evident acute infections, but in cases of doubtful infection they did provide confirmatory evidence of an antibody response in approximately one-third of patients culture-positive forPseudomonas aeruginosa.     

Cross-reaction between antibodies to the major epitope of Ro60 kD autoantigen and a homologous peptide of Coxsackie virus 2B protein

Coxsackie virus RNA has recently been detected in biopsy specimens of minor salivary glands from patients with primary Sjögren's Syndrome (pSS). A peptide derived from Coxsackie virus 2B protein (pepCoxs) presents 87% sequence homology with the 222–229 region of the major linear B‐cell epitope of Ro60 kD autoantigen (pep216–232). Synthetic peptides corresponding to pep216–232: ²¹⁶KALSVETEKLLKYLEAV²³² and pepCoxs: ³¹MVTSTITEKL LKNLVKI⁴⁷, were prepared. Sera from 42 patients with pSS and 43 patients with systemic lupus erythematosus (SLE) as well as sera from 27 healthy individuals (normal controls) and sera from 30 patients with rheumatoid arthritis (disease controls) were tested against the two homologous peptides. Twenty‐five percent of SLE sera and 33·3% of pSS sera reacted against pep216–232, whereas 28% of SLE sera and 37% of pSS sera recognized the pepCoxs. The sera reacting with pep216–232 were apparently the same as those reacting with pepCoxs. Normal sera and disease control sera presented only a limited reactivity against both peptides (ranging from 3·7% to 10%). Both peptides reacted more prominently with anti‐Ro/La (+) sera from pSS patients. Thus, pep216–232 was recognized by 17% of the anti‐Ro (+) sera and by 42% of the anti‐Ro/La (+) sera, whereas pepCoxs was recognized by 28·5% and 38% of the a‐Ro(+) and a‐Ro/La(+) sera, respectively. Purified anti‐pep216–232 antibodies readily reacted with both peptides while inhibition experiments revealed the specificity of this reaction. These results suggest a possible cross‐reaction between antibodies to the major linear B‐cell epitope of Ro60 kD autoantigen and the homologous pepCoxs in pSS patients. This cross‐reaction might potentially play a role in autoantibody formation and the perpetuation of the autoimmune response against Ro/SSA and La/SSB.     

Widespread immunoglobulin E-mediated hypersensitivity in the Sudan to the “green nimitti” midge,Cladotanytarsus lewisi (diptera: Chironomidae): I. Diagnosis by radioallergosorbent test

A radioallergosorbent test (RAST) has been developed for the diagnosis of hypersensitivity to “green nimitti” chironomid midges of the species Cladotanytarsus lewisi. There was a high percentage binding of ¹²⁵I-anti-IgE to the allergen particle complex by serum from subjects who were clinically hypersensitive, and the RAST was inhibited following incubations of allergic sera with an extract of the allergen. In 104 hypersensitive subjects (i.e., those with a positive skin test or clinical history of bronchial asthma, with or without rhinitis) and 21 controls, the RAST appeared to be specific and of diagnostic value: (1) The percentage binding was appreciably higher in 38 symptomatic individuals (group I) with strongly positive skin tests as compared with 36 patients with moderate skin reactivity (group II). (2) Seven symptomatic subjects with negative skin tests (group III) had a positive (>6% binding) green nimitti RAST. (3) Positive RASTs were demonstrable in 16 and of 17 patients with positive skin tests in whom the history was equivocal (group IV). (4) Six asymptomatic individuals with positive skin tests (group V) had low RAST values. (5) Six asymptomatic Sudanese controls with negative skin tests gave similar values to those of the group V subjects. (6) All of the sera from 15 nonatopic United Kingdom controls gave less than 6% binding of ¹²⁵I-anti-IgE. There was no statistical correlation between the concentrations of total IgE and the green nimitti RAST values. These results suggest that the RAST may be useful diagnostic test in green nimitti hypersensitivity and may also be of value in studies on the epidemiology and in the monitoring of treatment of this important and widespread allergy problem in the Sudan.     

A microplate adaptation of the solid-phase C1q immune complex assay

A method has been developed for the detection of C1q binding immune complexes in serum in which microculture plates are used as the solid-phase matrix for adsorption of C1q. This micromethod used only one-tenth of the amount of both C1q and [¹²⁵I]anti-human immunoglobulin per test and enabled 7 times as many samples to be tested in triplicate in comparison with the number performed in duplicate by the standard tube assay.¹²⁵I-labelled C1q studies showed that adsorption varied with the brand of microplate used, some types of plate binding up to 66% of the labelled material. This is considerably more than that bound by the polystyrene tubes generally used for this assay.The increased capacity of the method allowed the binding to plates coated with the same batch of C1q to be assessed at various times after storage for 8–10 weeks at both 4°C and ?70°C. A marked decrease in binding to C1q of aggregates of IgG was observed on storage for longer periods. Results with different batches of aggregated IgG which had been ultracentrifuged suggested that this may be used as an effective standard, stable on storage at ?70°C.A comparison with the standard tube method of aggregated IgG, normal control sera and sera from patients with SLE showed that the micromethod adaptation of the C1q solid-phase binding radioimmunoassay is more economical and easier to perform and does not impair accuracy or standardization.     

Microtitration agglutination for detection of Treponema hyodysenteriae antibody.

A microtitration agglutination test for the detection of Treponema hyodysenteriae antibody in swine and rabbit sera is described. The following methods provided the best test results: antigen produced from the spirochete after a culturing period of 36 to 44 h at 38 degrees C, washed antigen inactivated with 0.01% Merthiolate at 4 degrees C for 24 to 36 h, sera heated at 56 degrees C for 30 min, a diluent of phosphate-buffered saline (0.01 M, pH 7.2), and test results read macroscopically after 18 to 24 h of incubation at 38 degrees C. The test enabled detection of antibody against pathogenic T. hyodysenteriae with a high level of consistency and sensitivity. Sera against nonpathogenic T. hyodysenteriae produced low agglutinating titers (less than or equal to 1:8) when reacted against antigen from pathogenic isolates. Inactivated antigen remained stable for 7 to 10 days. Specificity of the reaction in the agglutination test was shown by absorption studies.     

Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.     

Enzyme-linked immunosorbent assay (ELISA) using antibody class capture for the detection of antitoxoplasma IgM.

Sera from 180 patients with suspected toxoplasmic lymphadenopathy were examined for antitoxoplasma IgM by an enzyme-linked immunosorbent assay (ELISA), using antibody class capture (ACCA). Of 82 positive ACCA results, 78 were confirmed by testing the IgM fractions of the sera, obtained by sucrose density gradient centrifugation (SDGC). The four positive results which could not be confirmed were all from patients with at least a year''s history of lymphadenopathy. Sera from 10 patients with low Sabin Feldman dye test (DT) titers gave positive ACCA results and subsequent specimens from them showed a rise in antibody concentration, confirming the diagnosis of acute toxoplasmosis. The antitoxoplasma IgM immunofluorescent antibody test (IgM-IFA) on whole serum was relatively insensitive and gave false-positive results with sera containing rheumatoid factor (RF) and antinuclear factor (ANF). There were no false-positive ACCA results with such sera, probably because the conjugates were prepared from F(ab'')2 fragments of antitoxoplasma serum. The ACCA proved to be sensitive, specific and easily automated enabling examination of large numbers of specimens.     

Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. I. Measurement of serum antibodies by radioimmunoassay.

Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed.     

The effect of ethnicity on the vascular responses to cold exposure of the extremities

Purpose

Cold injuries are more prevalent in individuals of African descent (AFD). Therefore, we investigated the effect of extremity cooling on skin blood flow (SkBF) and temperature (T _sk) between ethnic groups.

Methods

Thirty males [10 Caucasian (CAU), 10 Asian (ASN), 10 AFD] undertook three tests in 30 °C air whilst digit T _sk and SkBF were measured: (i) vasomotor threshold (VT) test—arm immersed in 35 °C water progressively cooled to 10 °C and rewarmed to 35 °C to identify vasoconstriction and vasodilatation; (ii) cold-induced vasodilatation (CIVD) test—hand immersed in 8 °C water for 30 min followed by spontaneous warming; (iii) cold sensitivity (CS) test—foot immersed in 15 °C water for 2 min followed by spontaneous warming. Cold sensory thresholds of the forearm and finger were also assessed.

Results

In the VT test, vasoconstriction and vasodilatation occurred at a warmer finger T _sk in AFD during cooling [21.2 (4.4) vs. 17.0 (3.1) °C, P = 0.034] and warming [22.0 (7.9) vs. 12.1 (4.1) °C, P = 0.002] compared with CAU. In the CIVD test, average SkBF during immersion was greater in CAU [42 (24) %] than ASN [25 (8) %, P = 0.036] and AFD [24 (13) %, P = 0.023]. Following immersion, SkBF was higher and rewarming faster in CAU [3.2 (0.4) °C min^?1] compared with AFD [2.5 (0.7) °C min^?1, P = 0.037], but neither group differed from ASN [3.0 (0.6) °C min^?1]. Responses to the CS test and cold sensory thresholds were similar between groups.

Conclusion

AFD experienced a more intense protracted finger vasoconstriction than CAU during hand immersion, whilst ASN experienced an intermediate response. This greater sensitivity to cold may explain why AFD are more susceptible to cold injuries.     

Identification of a 17-kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot technique.

Sera obtained from human patients, calves, sheep, and rabbits infected with Fasciola hepatica were tested by the Falcon assay screening test enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) techniques with Fasciola hepatica excretory-secretory antigens in order to evaluate their immunodiagnostic potential. The study included sera from 13 patients infected with F. hepatica or a history suggesting fascioliasis, 5 patients infected and treated with bithionol or praziquantel (3 were cured with bithionol), 10 patients infected with Schistosoma mansoni, 6 infected with Trichinella spiralis, and 13 controls and sera from calves, sheep, and rabbits with a primary F. hepatica infection. By FAST-ELISA with F. hepatica excretory-secretory antigens, the serum samples from fascioliasis patients gave the highest absorbance values, and the schistosomiasis patient sera gave intermediate values compared with a normal human serum control. Also by FAST-ELISA, the values for serum from patients with fascioliasis decreased steadily after cure, reaching normal levels 20 to 47 weeks postcure. In contrast, the serum from two patients who had been treated but were not yet cured had high levels of antibodies for up to 3 years of infection. By EITB, the serum samples from humans, rabbits, cattle, and sheep with fascioliasis recognized two antigenic polypeptides of 17 and 63 kilodaltons (kDa) in the form of sharp bands. For humans, this recognition lasted for at least 3 years of infection. Sera from individuals with schistosomiasis mansoni or trichinosis or from normal controls did not recognize the 17-kDa F. hepatica antigenic polypeptide. However, serum from one human with S. mansoni and one with T. spiralis infection has slight bands in the 63-kDa region, suggesting cross-reactivity. Reactivity to the 17-kDa polypeptide was absent in fascioliasis patients at 1 year postcure. Reactivity to the 63-kDa polypeptide was significantly diminished in fascioliasis patients at 1 year postcure. The sera from rabbits with a primary F. hepatica infection also recognized both the 17- and 63-kDa antigenic polypeptides by week 4 of infection. Reactivity to both antigens diminished significantly 6 weeks postcure and disappeared by 8 weeks postcure. The sera from infected cattle and sheep recognized these two antigenic polypeptides by week 8 of infection. These studies suggest that the 17-kDa F. hepatica excretory secretory antigen is an excellent candidate for the immunodiagnosis of acute and chronic fascioliasis. Purification of this antigen and its application to quantitative serologic tests will permit further analysis of its predictive value to evaluate cure.     

Serum levels of 25-hydroxy vitamin D in normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE)

Objective and design

The aim of the study was to examine possible variations in the levels of 25-hydroxy vitamin D [25-(OH)D] in sera from normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE).

Material

Serum concentrations of 25-(OH)D were measured in normal male and female Biozzi and C57BL/6 mice, at 3–4 weeks old and 8–10 weeks old, with a minimum of six animals/group. Levels of the vitamin were also determined in CR EAE-inoculated mice, and controls, during the course of the disease using a minimum of six animals/treatment.

Methods

Cardiac blood was collected from the groups of normal, control and CR EAE-sensitised mice and sera prepared, by centrifugation of clotted samples, and assayed for 25-(OH)D levels by chemiluminescence assay.

Results

Normal male and female Biozzi and C57BL/6 mice had significantly higher levels of 25-(OH)D at 8–10 weeks old compared to concentrations at 3–4 weeks of age (P < 0.005). Also, levels of the vitamin were significantly raised in C57BL/6 male and female mice compared to values in samples from corresponding Biozzi mice. In addition, the amounts of 25-(OH)D in sera from female Biozzi and C57BL/6 mice were significantly increased compared to strain and aged-matched male mice. The CR EAE mice with acute stage disease had significantly higher 25-(OH)D levels compared to controls (P < 0.005). Vitamin concentrations fell to within controls values with the progression of CR EAE.

Conclusions

Our preliminary studies have revealed marked differences between the amounts of 25-(OH)D in sera from Biozzi and C57BL/6 mice together with clear gender bias. The investigations also show significant, but selective changes, in levels of the vitamin during the course of CR EAE that are not always associated with the neurological disease state.