Asia I口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia I口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法。用纯化的Asia I型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合, 采用间接ELISA和有限稀释法筛选杂交瘤细胞。分别用ELISA、Western blotting检测mAb腹水的效价及其特异性。筛选到杂交瘤细胞2株, 腹水效价均在100×29以上; 以纯化后的Asia I型口蹄疫病毒vp2重组蛋白作为抗原, 利用Asia I型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia I型口蹄疫抗体。临床应用表明, 该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%。     

Purification and establishment of ELISA for vitellogenin of Japanese sardine (Sardinops melanostictus)

Two female-specific serum proteins (FSSPs) were detected immunologically in estradiol-treated Japanese sardine Sardinops melanostictus. The major FSSP was demonstrated to be a high molecular estradiol-inducible glycolipophosphoprotein with an immunological relation to a major yolk protein, and was suggested to be vitellogenin (VTG). VTG was purified using negative immunoaffinity chromatography. The isolated VTG was used for raising the specific antiserum against VTG. A homologous enzyme-linked immunosorbent assay (ELISA) was developed using the antiserum and the isolated VTG. The sensitivity range of the ELISA was 44 ng/ml to 2670 ng/ml of VTG concentration for the conditions used in our investigation.     

Use of immunoenzyme analysis for controlling the activity of antisera to insulin

Antisera to insulin were obtained from guinea pigs hyperimmunized with the use of Freund's adjuvant. To control the specific activity of antisera, the highly specific modern method of ELISA was used. Insulin was allowed to adsorb on the surface of the wells of polystyrene microplates, then consecutive dilutions of the tested antisera to insulin were placed into the wells. The insulin-antibody complexes thus obtained were detected by means of immunoperoxidase conjugate. Antibody titers in antisera obtained from different animals varied from 5 X 10(-3) to 10(-5). A good correlation between the results obtained by ELISA and by radioimmunoassay was observed.     

Development of mature BDNF‐specific sandwich ELISA

Mature brain‐derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme‐linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF‐specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti‐BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross‐reactivity to human recombinant NT‐3, NT‐4, nerve growth factor and negligible cross‐reactivity (0.99–4.99%) for proBDNF compared to commercial ELISA kits (33.18–91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of β‐site amyloid precursor protein cleaving enzyme 1 (BACE1)^?/? and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity.

     

Serodiagnosis of Mycoplasma pulmonis infection in mice and rats by an enzyme-linked immunosorbent assay

Murine antibody against Mycoplasma pulmonis (Mp) was detected sensitively and specifically in experimentally and naturally infected animals by an enzyme-linked immunosorbent assay (ELISA), using urease conjugated antimurine immunoglobulin. More than 98% of the experimentally infected mice and rats exhibited positive reaction in the ELISA two or more weeks after infection, and the titer remained for a prolonged period (up to one year) after infection. However, we failed to detect antibody in the sera of one-week-postinfected animals. Mice and rats from breeding colonies were tested with the ELISA and compared with isolation of Mp from the respiratory organs. Positive reactions were shown in the ELISA using the sera from 91% of the mice and 98% of the rats from which the organisms were isolated. Conversely, 97% of the mice and 78% of the rats among Mp-free animals showed negative results in the ELISA. The sensitivity and specificity of the complement fixation test, which has been used widely for serodiagnosis of Mp-infection, were apparently lower compared to those of the ELISA. From these results, the ELISA was found to be available for the serodiagnosis of Mp-infection in mice and rats.     

Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity.

Aspergillus fumigatus secretes an 18-kDa nonglycosylated IgE-binding protein. This protein was previously shown to be a ribotoxin, like alpha-sarcin and mitogillin. A 686-bp long A. fumigatus cDNA encoding an 18-kDa ribotoxin was cloned and expressed in Escherichia coli as a fusion protein with six adjacent histidines (rAsp f I/a). rAsp f I/a was purified to homogeneity by Ni(2+)-chelate affinity chromatography and refolded. The recombinant protein was enzymatically active resulting in the cleavage of 28S rRNA within a universally conserved region. rAsp f I/a was cytotoxic for EBV immortalized or PHA stimulated human PBMC. Furthermore, rAsp f I/a was recognized by murine mAb made against an 18-kDa ribotoxin. IgE of individuals allergic to A. fumigatus bound to rAsp f I/a as shown by ELISA, dot blots, and Western blots. rAsp f I/a elicited positive immediate type I skin reactions in individuals allergic to A. fumigatus but not in healthy control individuals. The results show that rAsp f I/a has similar functional characteristics when compared to the native 18-kDa ribotoxin. rAsp f I/a expressed in E. coli can therefore be used as a standardized Ag/allergen for serologic and clinical diagnosis of A. fumigatus-associated diseases.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Selective expression of a major allergen and cytotoxin, Asp f I, in Aspergillus fumigatus. Implications for the immunopathogenesis of Aspergillus-related diseases.

Asp f I is a major 18-kDa Aspergillus fumigatus allergen and a member of the mitogillin family of cytotoxins. The nucleotide sequence of the Asp f I gene was determined by sequencing polymerase chain reaction products amplified from A. fumigatus spore DNA. The entire 678-bp DNA includes an 81-bp leader sequence, preceding the N-terminal alanine codon, a 52-bp intron, and a 444-bp open reading frame, encoding a 149-amino acid protein (M(r) 16,899), which is 99% homologous to mitogillin from Aspergillus restrictus. A mAb-based ELISA was used to compare Asp f I levels in spores, mycelia, and culture filtrate, and to determine the kinetics of allergen production. Disrupted hyphae or spore extracts had a 1000-fold lower level of Asp f I than culture filtrate, suggesting that germination of spores and growth of the fungus are essential for allergen production. Asp f I levels in A. fumigatus and A. restrictus peaked at day 3 (0.87 to 12.1 micrograms/ml), however, the allergen was not detected in Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans cultures (< 1.5 ng/ml) on either days 3 or 8. Northern analysis confirmed that Asp f I mRNA was detected only in A. fumigatus and A. restrictus, but not in the other four Aspergillus spp. Asp f I-specific DNA was generated after polymerase chain reaction amplification of genomic mycelial DNA obtained from A. fumigatus and A. restrictus, but not from the other Aspergillus spp. The results show that Asp f I is selectively expressed in A. fumigatus, and suggest that this cytotoxin could be a specific virulence factor for A. fumigatus.     

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.     

Standardization of ELISA IgM and IgA for immunodiagnosis of human trichinosis

An ELISA test for trichinosis using as antigen a larvae soluble fraction from Trichinella spiralis was carried out for the detection of IgM and IgA specific antibodies in 45 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. All the patients had positive serology detected by precipitin test, bentonite floculation test, indirect hemagglutination test and ELISA IgG test. The cut-off value was determined using two criteria. Criterion A was determined in each plate, using three positive controls and two negative ones; the average of the negative controls and the weakest positive control, multiplied by a 1.2 factor was, considered the cut-off value. Criterion B was determined using the average plus three standard deviations from 64 apparently healthy persons serum samples. In both cases, three serum dilutions (1:10, 1:100 and 1:500) were used. The sensitivity of ELISA IgM was 100.0, 93.3 and 82.2% using serum dilutions of 1:10, 1:100 and 1:500 respectively (criterion A) and 100.0, 97.8 and 95.6% for the same dilutions (criterion B), whereas the values for ELISA IgA were: 100.0, 91.1 and 86.7% (criterion A) and 100.0, 100.0 and 91.1% (criterion B). In order to find out the specificity of ELISA IgM and ELISA IgA, additional 118 serum samples from individuals with other parasitoses, such as cysticercosis (18) hydatidosis (39), fascioliasis (12), toxocariasis (30), Chagas' disease (12) and individuals with non-specific eosinophilia (7), were also tested. ELISA IgM presented a specificity of 92.3, 93.4 and 97.3% (criterion A) and 96.2, 97.8 and 97.8% (criterion B) whereas the results for ELISA IgA were 97.8, 98.9 and 99.4% (criterion A) and 98.4% for the 1:10 and 1:100 dilutions and 100.0% for the 1:500 dilution (criterion B). The positive predictive values of ELISA IgM were 76.3, 77.8 and 88.1% (criterion A) and 86.5, 91.7 and 91.5% (criterion B) whereas the negative ones were 100.0, 98.3 and 95.7% (criterion A) and 100.0, 99.4 and 98.9% (criterion B). The positive predictive values of ELISA IgA were 91.8, 95.3 and 97.5% (criterion A) and 93.8, 93.8 and 100.0% (criterion B) whereas the negatives ones were: 100.0, 97.8 and 96.8% (criterion A) and 100.0, 100.0 and 97.8% (criterion B). The use of ELISA IgM and ELISA IgA in the immunodiagnosis of trichinosis is discussed.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals.

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.     

Design, synthesis, biological evaluation and molecular modeling of novel 1,3,4-oxadiazole derivatives based on Vanillic acid as potential immunosuppressive agents

In present study, a series of novel 1,3,4-oxadiazole derivatives have been designed, synthesized and purified. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of (1)H NMR, ESI-MS and elemental analyses. Besides, we evaluated their immunosuppressive activity. Most of these synthesized compounds were proved to have potent immunosuppressive activity and low toxicity. Among them, the bioassay results demonstrated that compounds 5c, 5n, 5p, 5o, 6f and 6g exhibited immunosuppressive activities with IC(50) concentration range from 1.25μM to 7.60 μM against the T cells, and the IC(50) of positive control (csa) is 2.12 μM. Moreover, all the title compounds were assayed for PI3K/AKT signaling pathway inhibition using the ELISA assay. We examined the compounds with potent inhibitory activities against IL-1, IL-6 and IL-10 released in ConA-simulated mouse lymph node cells. The results showed compounds 5o and 6f displayed the most potential biological activity against T cells (IC(50)=1.25 μM and 4.75 μM for T cells). The preliminary mechanism of compound 5o inhibition effects was also detected by flow cytometry (FCM). The results of apoptosis and ELISA assay demonstrated that the immunosuppressive activity of compounds 5o and 6f against T cells may be mediated by the inhibition of PI3Kγ/AKT signaling pathway. Molecular docking was performed to position compounds 5o and 6f into PI3Kγ binding site in order to indicate the potential target.     

Molecular cloning and the allergenic characterization of tropomyosin from Tyrophagus putrescentiae

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.     

Role of light in the in vivo and in vitro synthesis of spinach thioredoxin f

Upon continuous illumination of dark-grown spinach ( Spinacia oleracea L. cv. Winter Giant) seedlings, the thioredoxin f (Td f) content (ELISA) showed a steep rise, which can be evaluated after 3 and 36 h illumination as 3 times and 10 times the dark value, respectively. These figures correspond to 0.03% and 0.1% of total soluble protein, which means a higher biosynthetic rate for Td f compared to the average of total proteins in the earlier steps of plant development. After 40-50 h light the Td f level reached its highest value which remained stable for an additional 40 h and then decreased. Pulse-chase in vivo experiments with [³⁵S]-methionine also showed this sharp increase of Td f in the dark-light transition. From the pattern of decay of [³⁵S]-labelled Td f, a half-life of 7 h was determined for this chloroplast protein. In vitro translation experiments with poly(A)-mRNA isolated from illuminated young spinach seedlings, coupled to a wheat-germ synthesizing system, showed the appearance of a labelled fraction of ca 19 kDa molecular mass, recognizable by a specific Td f antiserum. When intact spinach chloroplasts were added to the translation assay medium, and then illuminated, the 19 kDa band disappeared, with a parallel increase of an internalized 13 kDa labelled polypeptide, also recognized by the Td f antiserum. These results are good evidence for a nuclear-coded synthesis of a Td f precursor, which travels through the chloroplast envelope, leaving the functional protein inside the organelle after the loss of a 6 kDa transit peptide.     

Use of a cellular ELISA for the detection of cell surface antigens.

A sensitive, convenient and inexpensive enzyme-linked immunosorbent assay (ELISA) is described for the detection and relative quantitation of cell surface antigens. The cells to be tested are rapidly glutaraldehyde-fixed to the wells of microtiter plates, which can be stored for later assay, if desired. Alternatively, adherent cells may be left unfixed. Following incubation with antibodies specific for the antigens of interest, an enzyme-linked second antibody conjugate is added, followed by the substrate for the enzyme, as in a conventional ELISA for soluble proteins. The method is a sensitive and accurate alternative to immunofluorescence flow cytometry for rapid and inexpensive screening of large numbers of cell samples.     

Serological Evidence of Widespread Circulation of West Nile Virus and Other Flaviviruses in Equines of the Pantanal,Brazil

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT₉₀) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.     

Clinical usefulness of urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori: a collaborative study in nine medical institutions in Japan

Background. A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit.
Materials and Methods. A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests.
Results. Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA.
Conclusions. The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.     

禽流感血凝素基因的原核表达及其在H9亚型诊断中的应用

根据H9N2亚型禽流感病毒血凝素基因序列设计并合成引物 ,从本室分离并保存的H9N2亚型禽流感病毒中扩增了预计约 16 83bp的血凝素基因 ,将此扩增产物克隆进pMD18-T载体 ,限制性酶切及序列测定后 ,进一步将其亚克隆到pGEX-KG中 ,与GST蛋白融合表达。SDS-PAGE和Western印迹表明缺失信号肽后的HA基因在大肠杆菌中获得了表达 ,表达产物具有免疫学活性 ,融合蛋白的分子量约为 90kD,位于包涵体中。包涵体经变性、复性处理 ,利用复性产物作为抗原包被酶标板建立了检测H9亚型禽流感抗体ELISA方法。结果表明应用HA重组蛋白作为诊断H9亚型禽流感抗原具有特异性强、敏感性高、重复性好的特点 ,可用于H9亚型禽流感抗体的检测。     

Rapid enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to Candida albicans

A rapid enzyme-linked immunosorbent assay (ELISA) where the performance time was shortened to 4h was compared with counter-immunoelectrophoresis (CIE) and a standard ELISA procedure for the detection of IgG antibodies to Candida albicans in 61 patients with suspected invasive candidosis. Using a C. albicans cytoplasmic antigen the rapid ELISA compared well with CIE and the standard ELISA. Seventeen sera that reacted with two concentrations of C. albicans antigen in CIE were also positive in both forms of ELISA. Four sera that were CIE-negative were positive in the standard ELISA and three were also positive in the rapid ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of different forms of candidosis.