Evaluation of a multiplex flow immunoassay versus conventional assays in detecting autoantibodies in systemic lupus erythematosus.

INTRODUCTION: Conventional diagnostic assays are being replaced with automated multiplex assays, but their performance needs to be evaluated. We compared a multiplex flow immunoassay with conventional techniques in the detection of antinuclear antibodies (ANAs) and antibodies to specific extractable nuclear antigens (ENAs) in serum samples from patients with systemic lupus erythematosus. METHODS: A total of 140 consecutive Chinese patients with systemic lupus erythematosus and 41 healthy controls were included. The automated BioPlex 2200 ANA Screen assay (Bio-Rad Laboratories, Hercules [CA], US) was compared with indirect immunofluorescence. In addition, use of BioPlex 2200 to detect anti-ENA antibodies was compared with in-house assays of countercurrent immunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA), and line blot. RESULTS: The sensitivity and specificity of BioPlex in detecting ANAs (91.4% and 95.1%, respectively) were comparable to those of indirect immunofluorescence (90.7% and 85.4%, respectively). Overall, BioPlex achieved the best agreement with ELISA in detecting anti-ENA antibodies: agreement was >90% for most antibody types (κ=0.79-0.94). In contrast, agreement was poorest with CIEP, ranging from 85.6% (κ=0.33) for anti-Sm antibodies to 93.9% (κ=0.88) for anti-Ro antibodies. Overall, BioPlex and ELISA had the highest sensitivity, whereas CIEP had the highest specificity. In terms of disease association, anti-Sm detected by CIEP had the best positive predictive value and specificity for lupus nephritis. CONCLUSIONS: In a local lupus cohort, BioPlex showed comparable sensitivity to indirect immunofluorescence in detecting ANAs and comparable performance to ELISA in detecting anti-ENA antibodies. However, CIEP was the best method in terms of disease specificity.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium]. / Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

External validation of recombinant antigens for serodiagnosis of machine operator's lung.

BACKGROUND: Machine operator's lung (MOL) is a hypersensitivity pneumonitis the diagnosis of which is difficult. Our laboratory previously developed an ELISA test using recombinant antigens from Mycobacterium immunogenum isolated in French plant. The objective was to validate the previous ELISA results with ten new suspected cases from Germany. METHODS: Two serological analyses were performed: ELISA with the six recombinant antigens, and electrosyneresis with crude antigens of M. immunogenum and three other main species isolated from contaminated metalworking fluids. RESULTS: The two recombinant antigens acyl-CoA dehydrogenase and dihydrolipoyl dehydrogenase, combined together, and electrosyneresis are useful in making the diagnosis regardless of the clinical and radiological data. Finally 9 out of the 10 suspected cases were declared as MOL. CONCLUSIONS: Despite the geographical distance, the crude and recombinant antigens produced to investigate the clustered French cases also proved to be useful in diagnosing the suspected cases in Germany.

ROLE OF SALMONELLA SEROLOGY A CENTURY AFTER THE WIDAL ERA.

Typhoid fever remains an important health burden in the developing world, whereas non-typhoid salmonelloses are one of the most common food-borne illnesses throughout the world and can be subjected to extra-intestinal complications. Culture is the gold standard for diagnosing a Salmonella infection. Serology can also provide evidence of infection. Serological methods for the diagnosis of Salmonella infections in humans and animals vary widely and can as well be used in epidemiologic studies to detect carriers, to assess infection rates, disease burden and vaccine responses. As with all serology, detecting antibody titers in Salmonella infections has its limits, mainly related to low sensitivity and specificity, high running costs, and antibody kinetics and peculiarities. Fast and more or less reliable immunoassays which detect Salmonella enterica serovar Typhi are commercially available. Veterinary and food sectors are well-provided with commercially tests for non-typhoid salmonellosis, while most immunoassays for non-typhoid human Salmonella diagnosis are developed and used in-house mainly for research or surveillance purposes. So far, there is no international consensus for the development of such serological tests for routine diagnostics. 119 years after the observations made by George Fernand Isidore Widal, this work intends to review and analyze the present state of facts and controversies in the field of Salmonella serology.

Caprine pleuropneumonia in Beetal goats [corrected].

Seroprevalence, clinical findings, and lesions of contagious caprine pleuropneumonia (CCPP) in Beetal goats were recorded during an outbreak. The overall seroprevalence of CCPP was 32.50%. Confirmation of Mycoplasma mycoides in serum was carried out using counter immunoelectrophoresis (CIE) technique. The highest CIE-positive cases were recorded in the older goats (51.72%) as compared to young ones. Nasal swabs collected from 39 goats showing respiratory signs were found positive for M. mycoides. The most consistent clinical findings were mild to severe cough, purulent nasal secretion, emaciation, dyspnea, increased respiration rate, and pyrexia. Mortality due to CCPP was 9.17%. Consolidation of lungs exhibited the highest frequency (100%), followed by alveolar exudation (90.90%) and pleural adhesion (72.72%). Among the microscopic lesions, septal peribronchiolar fibrosis exhibited the highest frequency (81.81%), followed by fibrinous pleuritis (63.63%) and peribronchiolar cuffing of mononuclear cells (54.54%) in lungs. From these results, it was concluded that CCPP under subtropical conditions has high prevalence in Beetal goats and leads to significant mortality.

Evaluation of a simple Dot-ELISA in comparison with countercurrent immunoelectrophoresis for diagnosis of human hydatidosis.

BACKGROUND: Cystic echinococcosis (CE) is caused by the larval stage of cestode parasite Echinococcus granulosus which has a worldwide distribution with variable geographic incidence. The diagnosis of CE is still problematic since the performance of the available serological assays are not reasonably satisfactory. The present study aimed to develop a specific and simple Dot-ELISA system and compare it with currently available countercurrent immunoelectrophoresis (CCIEP) technique for the diagnosis of human CE. METHODS: Serum samples were collected from thirty five pathologically confirmed CE patients, 25 healthy controls, and 12 non-CE patients. The hydatid cyst fluid (HCF) was aseptically obtained from sheep hydatid cysts. Anti-hydatid cyst antibodies in the serum were evaluated by Dot-ELISA and CCIEP. RESULTS: Findings of the study demonstrated a sensitivity of 100% and specificity of 89.1% for the Dot-ELISA system. Antibody was detected in 80% of patients by CCIEP while a few of non-CE patients and healthy controls had a positive reaction in this system. A sensitivity of 80% and specificity of 62% was calculated for this system. CONCLUSIONS: Dot-ELISA was found to be more sensitive and specific in detecting anti-hydatid cyst antibodies in CE patient in comparison with CCIEP.

Serological diagnosis of paracoccidioidomycosis using a Paracoccidioides spp. comercial antigen and the counterimmunoelectrophoresis method.

PURPOSE: In-house Paracoccidioides spp. antigens are commonly used in the serological diagnosis of paracoccidioidomycosis (PCM). The sensitivity and specificity of a commercial Paracoccidioides spp. antigen was assessed for PCM serological testing. METHOD: Counterimmunoelectrophoresis and double immunodiffusion were used to evaluate the Paracoccidioides ID Antigen® reagent in sera from PCM cases and patients with other diseases. RESULTS: All active PCM sera (n=24) were reactive using counterimmunoelectrophoresis (sensitivity = 100%), including 11 cases of infection by P. brasiliensis sensu stricto and one by P. americana. Fifteen (88%) out of 17 sera from patients on treatment or cured were reactive, including one case of P. lutzii infection. One to three bands of antigen-antibody precipitate were observed on the agarose gel, with a predominance of two to three bands in the test with untreated PCM sera or at the beginning of antifungal therapy. All sera from patients with histoplasmosis (n=7), aspergillosis (n=5), and other diseases (n=27) tested negative (specificity = 100%). The overall sensitivity and specificity using the commercial antigen and double diffusion test were 75% and 100%, respectively. CONCLUSION: The commercial antigen performed satisfactorily and may contribute to the dissemination of the use of serological tests for the PCM diagnosis.

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients.

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65% (DID), 79% (CIEP) and 95% (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89%, 99% and 100%, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.

Isolation of two bioactive diterpenic acids from Copaifera glycycarpa oleoresin by high-speed counter-current chromatography.

INTRODUCTION: Phytochemical and biological studies carried out on Copaifera species showed that their oleoresins and isolated compounds have various biological activities. OBJECTIVE: The aims of this work were (i) to analyse the Copaifera oleoresin by gas chromatography-mass spectrometry, (ii) to isolate the diterpenic acids from this oleoresin by high-speed countercurrent chromatography (HSCCC) and (iii) to determine the rhodamine 6G Pdr5p activity of these acids. METHODOLOGY: HSCCC was used for the preparative separation of the diterpenes. Spectroscopic methods were used to establish their identity. RESULTS: The gas chromatogram of the oleoresin showed approximately 30 compounds. The two major ones, kaur-16-en-18-oic and polyalthic acids, were isolated in high purity. Kaur-16-en-18-oic acid exhibited the highest rodomine 6G Pdr5p activity among the tested compounds. CONCLUSION: HSCCC was shown to be a quick and effective tool in the isolation and purification of diterpenes from Copaifera oleoresin. This is the first report on the use of HSCCC for the fractionation of an oleoresin from Copaifera and the isolation of diterpenes therein.

Accuracy of serological tests for diagnosis of chronic pulmonary aspergillosis: A systematic review and meta-analysis.

Chronic pulmonary aspergillosis (CPA) is a slow and progressive disease that develops in preexisting lung cavities of patients with tuberculosis sequelae, and it is associated with a high mortality rate. Serological tests such as double agar gel immunodiffusion test (DID) or counterimmunoelectrophoresis (CIE) test have been routinely used for CPA diagnosis in the absence of positive cultures. However, these tests have been replaced with enzyme-linked immunoassay (ELISA) and, a variety of methods. This systematic review compares ELISA accuracy to reference test (DID and/or CIE) accuracy in CPA diagnosis. It was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study was registered in PROSPERO under the registration number CRD42016046057. We searched the electronic databases MEDLINE (PubMed), EMBASE (Elsevier), LILACS (VHL), Cochrane library, and ISI Web of Science. Gray literature was researched using Google Scholar and conference abstracts. We included articles with patients or serum samples from patients with CPA who underwent two serological tests: ELISA (index test) and IDD and/or CIE (reference test). We used the test accuracy as a result. Original articles were considered without a restriction of date or language. The pooled sensitivity, specificity, and summary receiver operating characteristic curves were estimated. We included 14 studies in the review, but only four were included in the meta-analysis. The pooled sensitivities and specificities were 0.93 and 0.97 for the ELISA test. These values were 0.64 and 0.99 for the reference test (DID and/or CIE). Analyses of summary receiver operating characteristic curves yielded 0.99 for ELISA and 0.99 for the reference test (DID and/or CIE). Our meta-analysis suggests that the diagnostic accuracy of ELISA is greater than the reference tests (DID and/or CIE) for early CPA detection.

Dose response of black American mink to Aleutian mink disease virus.

INTRODUCTION: Aleutian mink disease virus (AMDV) causes a serious health problem for mink globally. The disease has no cure nor an effective vaccine and selection for tolerance using antibody titer is adopted by many mink farmers. The objective of this study was to investigate the effects of various doses of a local AMDV isolate on the response of black American mink to infection with AMDV. METHODS: Eight black American mink were each inoculated intranasally with 0.5 mL of eight serial 10-fold dilutions (100 to 10-7 ) of a 10% spleen homogenate containing a local AMDV isolate. Blood samples were collected on days 0, 20, 35, 56, 84, 140, and 196 postinoculation (dpi). Anti-AMDV antibodies and viral DNA were tested by counter-immunoelectrophoresis (CIEP) and PCR, respectively. Animals that were PCR or CIEP positive at 196 dpi (n = 41) were killed at 218 dpi, and samples of blood and seven organs were tested by CIEP and PCR. RESULTS: Antibody production persisted in all seroconverted mink until the termination of the experiment, whereas 71.1% of the mink showed short-lived viremia. Significant associations were observed between inoculum dose and the incidence of viremia until 84 dpi which disappeared thereafter, whereas associations between inoculum dose and the incidence of seropositive mink were significant on all sampling occasions. Antibody titer at 218 dpi significantly decreased with decreasing inoculum dose. AMDV DNA was detected in the bone marrow, lymph nodes, and spleen samples of almost all mink inoculated at every dose but was not detected in other organs of some mink. CONCLUSIONS: CIEP is more accurate than PCR for detecting AMDV infection in mink. Using antibody titer in naturally infected mink may not be accurate for the identification of tolerant mink.

Localization of sulfatoxygalactosylacylalkylglycerol at the surface of rat testicular germinal cells by immunocytochemical techniques: pH dependence of a nonimmunological reaction between immunoglobulin and germinal cells.

The synthesis of sulfatoxygalactosylacylalkylglycerol (SGG) is a marker of germinal cell differentiation during spermatogenesis. Antibodies raised against this lipid have been used to visualize SGG on the surfaces of rat spermatocytes and spermatids. An ionic interaction between SGG and immunoglobulin was shown to occur at physiological pH, resulting in high fluorescence backgrounds for control cells treated with nonimmune sera. Immunofluorescence was therefore performed at alkaline pH such that this interaction was much reduced or eliminated. A method was also developed to detect surface-bound complement fixed in the presence of anti-SGG. SGG was found to be mobile within the plane of the membrane, undergoing ligand-induced "patching" and occasional "capping." However, this phenomenon was independent of temperature.

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein.

Monospecific antibodies to bovine nasal cartilage proteoglycan monomer and link protein were used to demonstrate that immunologically related molecules are present in the bovine eye and associated tissues. With immunofluorescence microscopy, reactions for both proteoglycan and link protein were observed in the sclera, the anterior uveal tract, and the endoneurium of the optic nerve of the central nervous system. Antibody to bovine nasal cartilage proteoglycan also reacted with some connective tissue sheaths of rectus muscle and the perineurium of the optic nerve of the central nervous system. Antibody to proteoglycan purified from rat brain cross-reacted with bovine nasal cartilage proteoglycan, indicating structural similarities between these proteoglycans. ELISA studies and crossed immunoelectrophoresis demonstrated that purified dermatan sulphate proteoglycans isolated from bovine sclera did not react with these antibodies but that the antibody to cartilage proteoglycan reacted with other molecules extracted from sclera. Two molecular species resembling bovine nasal link protein in size and reactivity with antibody were also demonstrated in scleral extracts: the larger molecule was more common. Antibody to link protein reacted with the media of arterial vessels demonstrating the localization of arterial link protein described earlier. Tissues that were unstained for either molecule included the connective tissue stroma of the iris, retina, vitreous body, cornea, and the remainder of the uveal tract. These observations clearly demonstrate that tissues other than cartilage contain molecules that are immunologically related to cartilage-derived proteoglycans and link proteins.

Antibody seroprevalences against Peste des Petits Ruminants (PPR) virus in sheep and goats in Sudan.

Counter immnuo-electrophoresis (CIEP) and Competitive ELISA (C-ELISA) tests were employed for seroprevalence of Peste des Petits Ruminants (PPR) infection in Sudan. The result of both tests showed high prevalence of PPRV antibodies in sheep and goats sera collected from six different regions of Sudan. Of the 519 serum samples examined for the presence of PPRV antibodies 307(59.15%) were positive by CIEP while 263(50.67%) were positive by C-ELISA. CIEP technique was shown to be more sensitive than C-ELISA technique for detection of PPRV antibodies (Kappa statistics 0.259). C-ELISA allowed rapid, simple, specific, sensitive and differential sero-diagnosis of PPRV and RPV in sheep, goats and cattle. CIEP is, unlike competitive ELISA, is group-specific test and can not differentiate between PPR and RP infections. Despite its low specificity CIEP can be a useful indicative screening test for PPRV antibodies in flocks that neither been vaccinated nor otherwise exposed to PPR or RP virus. Results obtained suggest that CIEP, like the HI test, could be a useful screening test where it is not possible to use C-ELISA.

High values of CXCL10 serum levels in mixed cryoglobulinemia associated with hepatitis C infection.

OBJECTIVES: No study has evaluated circulating CXCL10 in patients with mixed cryoglobulinemia (MC) and hepatitis C virus (HCV) chronic infection. The aim of this study is to measure inteferon-inducible protein 10 (CXCL10/IP-10), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) (Th1 cytokines) in a series of cryoglobulinemic patients and to correlate this parameter to the clinical phenotype. METHODS: Serum CXCL10, IFN-gamma, and TNF-alpha were assayed in 102 patients with hepatitis C-associated cryoglobulinemia (MC + HCV), in 102 sex- and age-matched patients with type C chronic hepatitis without cryoglobulinemia (HCV+), and in 102 sex- and age-matched controls. RESULTS: Cryoglobulinemic patients showed significantly higher mean CXCL10 serum levels than controls (P < 0.0001) or HCV+ patients (P < 0.0001) (397 +/- 132 pg/mL, 92 +/- 53 pg/mL, 280 +/- 149 pg/mL, respectively). Moreover, CXCL10 was significantly increased in 30 cryoglobulinemic patients with active vasculitis compared to those without it (460 +/- 104 pg/mL vs 369 +/- 139 pg/mL, respectively; P < 0.001). Both groups of MC + HCV patients with or without active vasculitis had serum CXCL10 significantly higher than HCV+ patients (P < 0.001, P= 0.02, respectively). IFN-gamma levels were not significantly different in MC + HCV than in HCV+ patients or controls. Serum TNF-alpha levels were significantly higher in MC + HCV than in HCV+ patients or controls (median [interquartile range]: 12.0 [9.8], 5.7 [5.4], 1.3 [2.1] pg/mL, respectively; P < 0.0001). CONCLUSIONS: The study demonstrates high CXCL10 and TNF-alpha serum levels in patients with hepatitis C-associated cryoglobulinemia. Moreover, in MC + HCV patients, increased CXCL10 levels were significantly associated with the presence of active vasculitis.

Isotype switching and titer variation of anti-Ro/SSA antibodies over time in 100 patients with undifferentiated connective tissue disease (UCTD).

OBJECTIVE: To correlate the clinical course of the disease with the titer, the isotype profile and the switch of the anti-Ro/SSA antibodies in a cohort of patients affected by UCTD. METHODS: One hundred selected patients with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis (CIE), and affected by UCTD with a mean follow-up of 7.6 years (SD 4.8 yrs.), were studied. The titer of IgA, IgG and IgM anti-Ro/SSA antibodies was determined in two different sera, obtained at the time of diagnosis and at the last visit, by ELISA with Ro/SSA recombinant proteins as substrate. RESULTS: Thirty-five patients evolved from UCTD to a different connective tissue disease, while 65 showed a stable disease. Anti-Ro/SSA antibodies were detected in 91% and 97% of the patients, at baseline and during follow-up, respectively. IgG dominates the anti-Ro response. The titer of IgA, IgM and IgG anti-Ro/SSA did not differ significantly between the two groups of patients with UCTD. An increasing trend of IgG and IgA anti-Ro/SSA titer could be detected in patients evolving in primary Sjögren's Syndrome (pSS), but only the increase of IgG anti-Ro/SSA was significant (p=0.0235). CONCLUSION: IgG dominates the anti-Ro/SSA response in patients with UCTD. No substantial change of the antibody isotype against Ro/SSA peptides could be observed during follow-up. The titer of IgG anti-Ro/SSA significantly raised in the group of patients evolving in pSS.

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood.

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.

Clinical and serological differences between systemic lupus erythematosus patients with antibodies to Ro versus patients with antibodies to Ro and La.

Among 55 systemic lupus erythematosus patients having antibodies to Ro and/or La, two major groups were distinguished by titration of sera in counterimmunoelectrophoresis. The first group (30 patients) had antibodies to Ro alone. This was associated with a high incidence of antibodies to DNA (77%) and serious renal disease (53%). The second group (23 patients) had antibodies to Ro and La, and this was associated with a lower incidence of antibodies to DNA (30%) and a very low incidence of nephritis (9%). In this group a phenomenon of linkage of anti- Ro and anti-La titers was observed. Additionally two patients with only anti-La were found. Neither had clinically apparent renal disease. Thus, systemic lupus erythematosus patients with anti-Ro fall into two subgroups that differ considerably in their prevalence of anti-DNA and serious renal disease.

Studies of the human factor VIII/von Willebrand factor protein. III. Qualitative defects in von Willebrand's disease.

The Factor VIII/von Willebrand factor protein was characterized in two unrelated patients with von Willebrand's disease in whom procoagulant and Factor VIII/von Willebrand factor antigen levels were normal. In both patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis. In one patient the Factor VIII/von Willebrand factor protein eluted from Sepharose 4B in a position and distribution identical to normal with normal levels of procoagulant activity and antigen. However, the partially purified Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulant activity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulant activity are normal while the von Willebrand factor activity is deficient.

Carbohydrate of the factor VIII/von Willebrand factor in von Willebrand's disease.

We have examined the plasma Factor VIII/von Willebrand factor (FVIII/vWF) molecule from 16 patients with von Willebrand's disease, and have found no evidence of a significant decrease of carbohydrate content in 15 of these patients. FVIII/vWF was isolated by preparative counter immunoelectrophoresis directly from plasma using antibody to Factor VIII-related antigen, reduced in sodium dodecyl sulfate in the presence of urea, and electrophoresed in 5% polyacrylamide gels to separate the FVIII/vWF subunit from other proteins. Duplicate gels were stained by either the periodic acid-Schiff (PAS) reaction or by Coomassie Brilliant Blue G250. The ratio of Coomassie: PAS was determined by spectrophotometric scanning of the gels. Transferrin was used as an internal reference standard. The ratio for 23 normal individuals was 2.4+/-0.38 and the observed range was 1.8-3.8. 15 patients with von Willebrand's disease fell within this range. One patient independently reported as having decreased FVIII/vWF carbohydrate was also studied by this technique. A ratio of 6.8 was found, indicative of decreased, though not absent, carbohydrate. Cold insoluble globulin did not represent a significant contaminant in these analyses. 11 of the von Willebrand's disease patients with normal FVIII/vWF carbohydrate had abnormal crossed immunoelectrophoretic patterns characterized by absence of the less anodic forms of Factor VIII-related antigen. Four patients had normal patterns. These studies indicate that an absence or decrease of PAS reactive FVIII/vWF carbohydrate is not a consistent abnormality in von Willebrand's disease.