Specific Diagnostic Antigen Research Articles

Molecular characterization and serodiagnostic evaluation of the Echinococcus ortleppi recombinant glutaredoxin 1 protein for cystic echinococcosis in buffalo (Bubalus bubalis)

Cystic echinococcosis (CE), caused by the metacestode of Echinococcus granulosus sensu lato (s.l.), adversely affects the physiology of the vital organs in which they grow. Condemnation of meat causes substantial economic loss to the livestock industry. Conventionally the infection is detected by necropsy as serological diagnosis of the infection in livestock is ambiguous. Identification of specific diagnostic antigens would be a substitute for the cyst fluid antigens which lack adequate diagnostic sensitivity and specificity. BLAST analysis supported by the negligible pairwise nucleotide distance of the 389 nt COX1, 489 nt NAD1, and 425 nt ITS1 with the related sequences of E. ortleppi ascertained the association of E. ortleppi with CE in buffaloes. Given the extensive distribution of glutaredoxin 1 in every developmental stage of Echinococcus granulosus s.l that makes it an ideal serodiagnostic antigen for CE, we expressed the 14 kDa E. ortleppi glutaredoxin 1 (rEoGrx1) protein in E. coli BL21 (DE3) and tested a total of 225 sera samples, including 126 sera samples from the necropsy-positive buffalo, by the rEoGrx1 IgG-ELISA. The ELISA could detect a total of 82/126 sera samples as positive. The diagnostic sensitivity and specificity of the rEoGrx1 IgG-ELISA were 65.1 % and 51.5 %, respectively. The protein showed serological cross-reaction against Fasciola gigantica, Toxoplasma gondii, and Sarcocystis sp. The in silico bioinformatics analysis of the E. ortleppi, F. gigantica, and T. gondii glutaredoxin sequences revealed fully conserved amino acids at positions 11 and 21, the substitution of conserved amino acids at positions 14 and 6, and semi-conserved substitutions at positions 3 and 4, respectively. The findings partly explain the molecular basis of the serological cross-reactivity of the protein.

Study on antigenic protein Omp2b in combination with Omp31 and BP26 for serological detection of human brucellosis

BackgroundBrucellosis is a very common zoonosis in certain localized areas worldwide, with a high prevalence in most developing countries. The detection of brucellosis still faces many challenges such as the need for more sensitive and specific diagnostic antigens. MethodsTo evaluate the efficacy of Brucella outer membrane proteins (Omps) Omp2b in combination with omp31 and BP26 as diagnostic antigens for the serological detection of human brucellosis, these proteins were prepared by a prokaryotic expression system. Human brucellosis-positive and-negative sera were collected, and the detection effects of the diagnostic antigens were evaluated using an established indirect ELISA (iELISA) method. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC), true positives, true negatives, false positives, false negatives, accuracy, positive predictive value, negative predictive value, analytical specificity, and sensitivity were obtained to evaluate the effectiveness of Omp2b and antigen combinations. ResultsThe iELISA results showed that the AUC of the antigenic proteins was 0.9100, 0.9387, 0.9343, and 0.9448, respectively, and that the combination of Omp31 and BP26 improved the accuracy and was superior to that of Omp2b alone. Analysis at the determined cut-off values showed that the analytical sensitivity of the assay was 0.8739 (95% CI:0.7974–0.9293) and the analytical specificity was 0.8539 (95% CI:0.7632–0.9199) when using Omp2b alone and 0.8649 when using the combination of Omp2b + BP26 (95% CI:0.7869–0.9223) with an analytical specificity of 0.9213 (95% CI:0.8446–0.9678) and 0.8468 (95% CI:0.7662–0.9082) and an analytical sensitivity of 0.9101 (95% CI:0.8305–0.9604). When Omp2b + Omp31 + BP26 was combined, the analytical sensitivity and specificity were 0.8559 (95% CI:0.7765–0.9153) and 0.9326 (95% CI:0.8590–0.9749), respectively. Protein antigens, including antigen combinations, did not cross-react with Yersinia enterocolitica O9 and E. coli O157: H7, indicating that their specificity was better than that of lipopolysaccharide (LPS). ConclusionsCompared with individual Omp2b, antigen combinations improved the effectiveness in detecting brucellosis, but were still not as effective as LPS antigen. Omp2b, combined with Omp31 and BP26 as diagnostic antigens, can be used to detect human brucellosis.

TsPKA-r: a potential immunodiagnostic antigen for the detection of porcine cysticercosis

Cysticercosis, caused by metacestodes of Taenia solium, has a significant soci-economic impact and is of considerable importance in public health. However, there are no specific diagnostic antigens to distinguish between T. solim and Taenia hydatigena. In the present study, cAMP-dependent protein kinase regulatory subunit (TsPKA-r), an excretory/secretary (ES) antigen of T. solium, was used to establish a specific and sensitive diagnostic tool for detection of porcine cysticercosis. The full-length sequence encoding TsPKA-r was amplified by PCR, sequenced and then identified by bioinformatics. The fusion protein with 6×His-tags was expressed in E. coli, purified by Ni Sepharose™ 6 Fast Flow and used to test reactionogenicity by immunoblotting. TsPKA-r based indirect enzyme-linked immunosorbent assays (iELISA) showed good performance in recognition of sera of pigs experimentally infected with T. solium metacestodes, with 93.88% sensitivity and 96.40% specificity. There were no cross-reactions against the sera from pigs experimentally infected with T. hydatigena, Toxoplasma gondii or Trichinella spiralis. These results indicate that the TsPKA-r is a promising immunodiagnostic antigen for detection of porcine cysticercosis.

Kinetics ofFasciola giganticaAntibody Response in Naturally Infected Bovine Ensuing Levamisole and Oxyclozanide Combination Therapy

A study was conducted to determine the kinetics of Fasciolagigantica infection induced antibody response against oxyclozanide-levamisole in naturally infected bovine of South Gujarat, India. Faecal analysis on before, 1st, 4th, 8th, 11th and 37th days post treatment (DPT) recorded 67.14, 34.29, 5.71, 0 and 0% parasites infected bovines. Either parasite(s) positive indigenous cattle or buffalo become parasite(s) free by 8th DPT but a lag period of 4 days had been recorded in the exotic cattle. The trematode parasites infected animals take more time to clear the infection with the used drugs, it's become 100% negative on 11th DPT while the nematodes parasites infected animals successfully clear the infection by 4th DPT and maintain it until the end of experiment. The 5 animals which showed reactivity in dipstick ELISA test following natural infection with F. gigantica using parasites specific diagnostic antigen, cathepsin L cysteine proteinase (CP), was subjected to indirect plate ELISA assay, the mean anti-CP antibody titer was recorded at its maximum at pretreatment in treated group positive animal when probed with CP antigen. Thereafter, a declining trend of antibody response was recorded in these naturally infected bovines which reached to undetectable level on 90th DPT in response to anthelmintic therapy while untreated animals (negative and positive animals) showed almost similar pattern of antibody response during the experiment. A significant decrease in % eosinophilia count was also observed at 37th DPT in response to treatment.

Cloning and expression and preliminary antigenicity identification for the diagnostic antigen of hepatitis E virus

To clone, express and purify thioredoxin (named as N5), a specific diagnostic antigen of hepatitis E virus (HEV), and to initially evaluate its antigenicity and serological test performance. Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank, the codon was optimized by the Escherichia coli codon preference, inserted it into prokaryotic expression vector M48 following total gene synthesization, and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX). Fusion protein was purified in affinity chromatography, evaluating its antigenicity with Western blot technology, then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests. The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive, showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA, testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people, with the sensitivity and specificity of 95% (38/40) and 90% (36/40) respectively. Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established, and soluble fusion protein N5 was obtained with high expression and strong antigenicity, promising in its future applications.

Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics

BackgroundThe excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae (ML) come mainly from the excretory granules of the stichosome and the cuticles (membrane proteins), are directly exposed to the host’s immune system, and are the main target antigens, which induce the immune responses. Although the ES proteins are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage are the false negative results during the early stage of infection. The aim of this study was to identify early specific diagnostic antigens from the main components of T. spiralis muscle larval ES proteins.MethodsTwo-dimensional electrophoresis (2-DE) combined with Western blot were used to screen the early diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The protein spots recognized by the sera from BALB/c mice infected with T. spiralis at 18 days post-infection (dpi) were identified by MALDI-TOF/TOF-MS and putatively annotated using GO terms obtained from the InterPro databases.ResultsThe ES proteins were analyzed by 2-DE, and more than 33 protein spots were detected with molecular weight varying from 40 to 60 kDa and isoelectric point (pI) from 4 to 7. When probed with the sera from infected mice at 18 dpi, 21 protein spots were recognized and then identified, and they were characterized to correlate with five different proteins of T. spiralis, including two serine proteases, one deoxyribonuclease (DNase) II, and two kinds of trypsin. The five proteins were functionally categorized into molecular function and biological process according to GO hierarchy.Conclusions2-DE and Western blot combined with MALDI-TOF/TOF-MS were used to screen the diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The five proteins of T. spiralis identified (two serine proteases, DNase II and two kinds of trypsin) might be the early specific diagnostic antigens of trichinellosis.

Proteomic Analysis ofTrichinella spiralisMuscle Larval Excretory-Secretory Proteins Recognized by Early Infection Sera

Although the excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage is the false negative results during the early stage of infection and cross-reaction of their main components (43, 45, 49, and 53 kDa) with sera of patients with other helminthiasis. The aim of this study was to identify early specific diagnostic antigens in T. spiralis ES proteins with 30–40 kDa. The ES proteins were analyzed by two-dimensional electrophoresis (2-DE), and a total of approximately 150 proteins spots were detected with isoelectric point (pI) varying from 4 to 7 and molecular weight from 14 to 66 kDa. When probed with sera from infected mice at 18 days postinfection, ten protein spots with molecular weight of 30–40 kDa were recognized and identified by MALDI-TOF/TOF-MS. All of ten spots were successfully identified and characterized to correlate with five different proteins, including two potential serine proteases, one antigen targeted by protective antibodies, one deoxyribonuclease (DNase) II, and one conserved hypothetical protein. These proteins might be the early specific diagnostic antigens for trichinellosis.

Escherichia coli–expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen

BackgroundThe Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies.MethodsA deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels.ResultsIn-frame deletion of three hydrophobic regions, spanning amino acid residues 1–43, 519–538 and 676–706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses.ConclusionsThis study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.

Molecular and Immunological Characterization of a Novel 32-kDa Secreted Protein of Babesia microti

A cDNA encoding the Babesia microti 32-kDa protein was identified by serological immunoscreening of a cDNA expression library and designated as BmP32. The full length of BmP32 contains an open reading frame of 918 base pairs consisting of 306 amino acids having a significant homology with B. microti secreted antigen 1. Antiserum raised against recombinant protein (rBmP32) specifically reacted with a 32-kDa native protein of the parasite lysate using western blot analysis. The indirect immunofluorescent antibody test showed a preferable localization of BmP32 in the cytoplasm of the intra- and extracellular parasites. Moreover, BmP32 was secreted in the cytosol of infected erythrocytes, especially during the peak parasitemia and the recovery phase of the infection. Next, the antigenicity of rBmP32 was examined by an enzyme-linked immunosorbent assay (ELISA) and sera from mice experimentally infected with either B. microti or closely related parasites. ELISA was highly specific and sensitive when used for the detection of B. microti antibody in a mouse model. Furthermore, mice immunized with rBmP32 emulsified with Freund's adjuvant were not significantly protected against challenge infection with B. microt i. However, high antibody titer was detected just before the challenge infection. Our data suggest that rBmP32 may be a specific diagnostic antigen but not a subunit vaccine.

Immunoaffinity chromatographic analysis for purification of specific diagnostic antigens of Paramphistomum epiclitum

Polypeptide profile of somatic antigen of Paramphistomum epiclitum (PSAg) and Gastrothylax crumenifer (GSAg) was studied by SDS-PAGE. PSAg and GSAg showed 14 and 19 polypeptides in the range of 14.9-95.5 and 13.7-129.6kDa with six common polypeptides of mol wt 16.8, 21.8, 23.7, 35.5, 43.4 and 70.8kDa. P. epiclitum experimentally infected sheep sera were used for identification of specific immuno-dominant peptide in the range of 37-40kDa against P. epiclitum by western blotting. Hyperimmune sera (HIS) was raised in rabbit against the identified polypeptide, IgG was separated from HIS and an immunoaffinity column was constructed with a binding percentage of 83.74 of IgG with CNBr activated Sepharose 4B. Purification of somatic antigen (PSAg) was done with immunoaffinity chromatography and 37-40kDa protein antigen was isolated in pure form with recovery percentage of 2.97%. This purified fraction of somatic antigen can be used as a candidate antigen for development of serological assay for early diagnosis of paramphistomosis among livestock.

Molecular characterization of a novel 32-kDa merozoite antigen ofBabesia gibsoniwith a better diagnostic performance by enzyme-linked immunosorbent assay

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

Specific diagnostic antigens of Echinococcus granulosus detected by western blot.

A western blot assay was performed for the detection of Echinococcus granulosus specific antigens useful for the diagnostic of hydatic disease. 191 sera were tested, 105 coming from patients with different localizations of hydatic cysts and 86 from persons either healthy or presenting other diseases. 48 different antigenic bands were detected using sera from patients with hydatidosis. A 35 kDa antigen co-migrating with a band labeled by a McAb specific of antigen 5 was recognized in western blot by only 68% of the sera able to precipitate antigen 5 in immunoelectrophoresis. A 8 kDa antigen corresponding to the specific E. granulosus antigen already described has been recognized by 80% of the sera coming from patients with hydatidosis and not by the 86 control sera. Bands of 21, 30, and 92 kDa appeared also specific and were recognized by at least 50% of tested sera. These antigens appeared unrelated one to each other. 103 out of the 105 sera from patients with hydatidosis were able to recognize at least one of the 8, 21, 30, 35 or 92 kDa specific antigens. The present results suggest that western blot could be useful for the diagnosis of hydatidosis as far as the criteria of positivity is based on the recognition of at least one of the major specific antigens.

A simplified method for the fractionation ofGnathostoma-specific antigens for serodiagnosis of human gnathostomosis

Specific immunoreactive components present in crude somatic extract and in excretory-secretory (ES) products of Gnathostoma spinigerum advanced third-stage larvae (L3) were identified by Western blotting and their diagnostic potential evaluated by indirect ELISA. Although both crude antigen preparations were highly complex, the ES antigen gave a more satisfactory diagnostic result. Most G. spinigerum specific components present in the somatic and ES preparations had molecular weights below 29 kD and were not glycosylated, judging from the concanavalin A staining pattern. Specific diagnostic antigens were prepared by subjecting the crude preparations to SDS-PAGE. Low molecular weight components were identified and electroeluted from the gel. Excess SDS was removed by the use of an ion retardation resin. The antigens obtained by this relatively simple procedure were found to be highly specific for G. spinigerum. Sensitivity, specificity, and positive and negative predictive values for the assay using the fractionated somatic antigen were 100%.

Diagnosis of rickettsial diseases: A perspective

Rickettsioses have nonspecific clinical manifestations, making them difficult to diagnose in a clinical setting. Laboratory testing is usually needed to confirm the diagnosis. Rickettsial isolation is a sensitive and specific diagnostic technique, but the hazards associated with handling pathogenic rickettsiae usually preclude isolation attempts in most laboratories. Rickettsiae can also be detected in infected tissues by fluorescein-labeled antisera or by immunoperoxidase staining, but these techniques lack sensitivity, except when applied to postmortem tissue specimens. However, rickettsial DNA can be detected in acute phase blood specimens by polymerase chain reaction (PCR) technology, and this technique offers the prospect of prompt diagnosis and treatment. Serologic testing remains the most frequently used approach to diagnosis, although antibody tests usually fail to identify rickettsioses early enough to affect the management of individual patients. Available serologic techniques vary considerably in their sensitivity and specificity. Enzyme-linked immunosorbent assays (ELISA) are extremely sensitive, but the general unavailability of specific diagnostic antigens reduces the specificity of this and other serologic techniques. Molecular characterization of rickettsial antigens may soon allow the production of peptide antigens that are specific for each species and could maximize the specificity of test results. No diagnostic technique has any value unless it is applied successfully to the appropriate patient population. Improved surveillance of rickettsial diseases is urgently needed to identify specific areas in which rickettsioses are endemic. Such surveillance data would promote awareness of rickettsioses among local physicians and increase the probability that individual patients with rickettsioses would be identified promptly and receive appropriate therapy early in the course of their illness.

Isolation and purification of a diagnostic antigen for bovine cysticercosis by hydrophobic chromatography

An ammonium sulfate fraction of Taenia hydatigena cyst fluid (ThFAS) was further fractionated by hydrophobic interaction chromatography, using alkylagarose and ω-amino alkylagarose columns, in an effort to isolate and purify a specific diagnostic antigen in the ThFAS preparation. The < 12kDa antigen was found to have an affinity for immobilized alkanes with chain length of six carbons or greater. The antigen was recovered in an ethylene glycol eluate from a hexylagarose column then analyzed by Western blot; it reacted with bovine and human cysticercosis infection sera and with specific monoclonal antibodies but not with control sera or fascioliasis infection sera. When the eluate was used as coating antigen in a plate ELISA assay no false positive reactions were seen in sera from cattle infected with Fasciola hepatica; false positive reactions were observed for the unfractionated ThFAS antigen preparation.

Surface antigens of male worms and microfilariae of Onchocerca gibsoni

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyi sulphate (SDS)-polyaerylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelied surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity ofmicrofilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

The use of cell-free antigens ofParacoccidioides brasiliensisin serological tests

A simple and rapid method for extracting specific cell-free antigens (CFA) from the yeast form of Paracoccidioides brasiliensis grown on agar slants was developed. Extracts were analysed mainly by immunodiffusion (ID) tests, for the immunodiagnosis of paracoccidioidomycosis. Extracts were obtained from 2 to 7-day-old cultures. The extract obtained after the third day, which contained 200-300 micrograms of protein/ml, gave the best ID reactions, with 100% sensitivity and specificity. The main precipitation band was intense and showed total identity with that formed by the specific 43 kDa glycoprotein. Other tests such as counterimmunoelectrophoresis and immunoelectrophoresis were also used successfully with CFA. The 3-day CFA extract showed a pattern of polypeptides in the 10-110 kDa range after SDS-polyacrylamide gel electrophoresis. Five components of 20, 43, 45, 55 and 70 kDa reacted on immunoblots with sera from patients with active chronic paracoccidioidomycosis. The specific diagnostic antigen of 43 kDa always predominated in all CFA preparations. The present method is thus very useful for the rapid production of an antigenic extract which can be readily characterized and used in the serodiagnosis of paracoccidioidomycosis.

Antigens of Histoplasma capsulatum. I. Serologic differences between histoplasmins derived from albino and brown filamentous isolates.

Recent studies indicate that Histoplasma capsulatum may be separated culturally into two distinct morphologic types, albino (A) and brown (B), at the time of primary isolation from clinical materials, and that the B variety appears to be the more virulent for the experimental animal. It is probable that the diagnostic antigens presently used may have been derived from the less virulent A filamentous form since A displaces B unless separated at the time of primary isolation. The antigens of histoplasmins prepared from primary isolates of A and B were compared with each other, and with antigens from standard HKC-43 histoplasmin, using DEAE-cellulose, gel-column chromatography and agar-precipitin analysis. Preliminary studies of the chromatography profiles did not reveal distinct qualitative or even quanitative differences between A- and B-derived histoplasmins. However, the gel-precipitin analyses verify the presence of an antigen unique to the B-derived histoplasmin. Since the B antigen appears also to be strain specific, these results suggest the need to develop specific diagnostic antigens derived from the B as well as the A varieties for the serologic study of histoplasmosis.

Factors influencing the formaldehyde treatment of viral antigens.

An investigation has been made of the use of formaldehyde for the preparation of stable non-infective viral antigens of influenza (PR8) and mumps (Enders). The stability of the antigen to formaldehyde treatment was influenced by the pH, the suspending medium of the virus, the formaldehyde concentration, and the duration and temperature of treatment. The following procedure was found to be the most satisfactory. The supernatant liquid of allantoic fluid which had been frozen and allowed to thaw slowly was centrifuged and the virus was resuspended in isotonic phosphate buffer at pH 6.0. A hemagglutination titer of about 10,000 was attained before the process of treatment was started. This viral antigen was filtered through coarse filter paper and treated with 0.1% formaldehyde at 45 °C. for 12 hr. The excess formaldehyde was neutralized by the addition of 0.25 cc. of 30% dibasic ammonium phosphate per 10 cc. of antigen. After 30 min. at room temperature, 5% (w/v) of arginine was added and the antigen was lyophilized in ampoules. These antigens have been found to be non-infective, satisfactory in the complement-fixation and hemagglutination-inhibition tests as specific diagnostic antigens, and show greater stability than liquid viral antigens.