Analysis of the human antibody response to outer surface protein C (OspC) of Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii.

The aim of this study was to determine by Western blotting (WB) the prevalence of anti-outer surface protein C (OspC) IgM and IgG antibodies in patients with Lyme borreliosis according to each of the three genospecies of Borrelia burgdorferi sensu lato. Strains of B. burgdorferi sensu stricto (MUL), B. garinii (DK 6), and B. afzelii (DK 26) served as antigen, all of which expressed abundant OspC. We examined sera from 117 patients with untreated early and late Lyme borreliosis, as well as from 100 blood donors and 29 patients with syphilis. WB results were compared with the B. burgdorferi flagellum enzyme-linked immunosorbent assay (ELISA) data. OspC from B. burgdorferi sensu stricto showed the lowest diagnostic sensitivity. OspC from B. garinii and B. afzelii performed almost identically in erythema migrans, with an IgM positive rate of 36% versus 34%, whereas OspC from B. garinii performed best in neuroborreliosis (60% versus 44%). The anti-OspC IgG response was less prominent than the IgM response and was infrequent in the late stages of the disease (0-20%). The benefit of combining the evaluation of anti-OspC responses with all three species was limited. The overall diagnostic sensitivity of WB anti-B. garinii OspC evaluation was, in the early stages of the disease, comparable to the results obtained using the flagellum ELISA. In erythema migrans and neuroborreliosis, the addition of anti-OspC IgM to the flagellum ELISA increased the sensitivity by 15% and 10%, respectively. It can, therefore, be concluded that OspC from B. garinii is a suitable OspC test antigen, and that supplementary use of OspC from other species adds little to the overall diagnostic sensitivity. An ELISA based on B. garinii OspC and native flagella seems currently the most promising concept for a future antibody test in early Lyme borreliosis.

Scientific dishonesty and good scientific practice.

Scientific dishonesty has been the subject of much public interest in recent years. Although the problem has had a low profile in Denmark, there is no reason to believe that it is non-existent. Several preconditions known to be important prevail here as well as in other countries, such as pressure to publish and severe competition for research grants and senior academic positions. The Danish Medical Research Council (DMRC) decided to respond to this problem by preparing a report on scientific dishonesty with suggestions to the research institutions on rules for good scientific practice and procedures for investigation of suspected dishonesty. To this end, an investigatory system was suggested. The system should consist of two regional committees and one national committee. They should be headed by high court judges and experienced health sciences researchers as members. The committees will investigate cases reported to them and conclude on whether dishonesty has been established and on whether the scientific work should be retracted. Sanctions shall remain the task of the institutions. Preventive measures comprise open access to and a long storage period for scientific data.

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein.

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.

Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12.

A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the 'common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

Counter current line deflection immunoelectrophoresis. A technique for the quantification of antibodies.

Combining the electrophoretic principles of counter current immunoelectrophoresis and deflection of line precipitates in line immunoelectrophoresis provides a new technique for quantitative determination of antibodies against specific antigens (CCLD electrophoresis), even if no purified antigen or monospecific antibodies are available for construction of the detection system. We have used the method for quantitative determination of antibodies against the flagellum of Treponema phagedenis biotype Reiter and compared the diagnostic potential of this method in the diagnosis of syphilis with an ELISA method for the quantification of IgG antibodies against the flagellum. The CCLD electrophoresis could be optimized to a diagnostic performance very similar to that achieved using the ELISA method.

Computer controlled affinity chromatographical purification of soluble Plasmodium falciparum antigens from supernatants of in vitro cultures.

Affinity chromatographic procedures are difficult to scale up from the analytical to the preparative level when the ligand used for purification is a limiting factor. A versatile, computer-controlled affinity chromatographic system is described which permits automatic repetition of the purification process and sophisticated control functions based on the ultra-violet absorbance of fluid passing through the affinity column. The system has been used for automation and scaling up of the purification of Plasmodium falciparum exoantigens.

Purified flagella from Treponema phagedenis biotype Reiter does not induce protective immunity against experimental syphilis in rabbits.

Thirteen rabbits were immunized three times at weekly intervals with 50 micrograms of flagella purified from Treponema phagedenis biotype Reiter. Fourteen rabbits were inoculated in the same way with a placebo preparation. Rabbits immunized with the flagella developed an immune response to the flagella but showed no statistically significant prolongation of incubation time or diminution of lesion severity when challenged intradermally with 4 X 10(3) Treponema pallidum organisms.

Immunological cross-reaction between antigen Tp-4 of Treponema pallidum and an antigen common to a wide range of bacteria.

By electro-immunoprecipitation methods it was shown that antigen Tp-4 of Treponema pallidum, antigen TR-c of the Reiter treponeme and 'Common Antigen' of Pseudomonas aeruginosa are immunologically cross-reactive. This finding establishes a taxonomic relationship between T. pallidum and a wide range of bacteria. The cross-reactivity explains some false positive reactions in serodiagnostic treponemal tests for syphilis. The cross-reactivity may also play a role in a possible 'normal' immunity against infection with T. pallidum induced by antigens by the normal human bacterial flora.

Determination of pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3) in gastric juice by rocket immunoelectrophoresis.

Antisera were raised in rabbits against chromatographically purified preparations of pepsin and gastricsin. With these antisera the contents of pepsin and gastricsin in gastric juice were determined by rocket immunoelectrophoresis. The potential content of pepsin and gastricsin of a secondary standard of gastric mucosal extract was calibrated against the chromatographically purified enzymes. This secondary standard was used for routine analyses. The intra-assay and between-assay precision was 3-4% and 6-9%, respectively. Ten healthy volunteers underwent a standard pentagastrin test. The amounts of pepsin and gastricsin determined by rocket immunoelectrophoresis corresponded to the amounts observed by ion exchange chromatography of gastric juice. After stimulation with pentagastrin the secretion of both pepsin and gastricsin was increased about 10 times.

Ribonucleic acid antigen from the Reiter treponeme used in an ELISA for antibodies in syphilis.

Purified RNA from Treponema phagedenis biotype Reiter was used as antigen in an enzyme-linked immunosorbent assay (ELISA) for IgG antibodies in syphilis. The RNA ELISA was compared with the fluorescent treponemal antibody absorption test (FTA-ABS), the Treponema pallidum immobilization test (TPI), and with two ELISAs using purified flagellum from the Reiter treponeme as antigen (flagellum ELISA), and sonified Reiter treponeme culture as antigen (sonicate ELISA). A total of 729 sera from patients with and without syphilis were studied. The RNA ELISA had a lower sensitivity (P less than 0.01) in primary syphilis than the flagellum ELISA, the sonicate ELISA, and the FTA-ABS. In treated syphilis the RNA ELISA was also less sensitive than the TPI (P less than 0.01).

Purification of TR-b, a Reiter treponeme protein antigen precipitating with antibodies in human syphilitic sera.

TR-b is a Reiter treponeme antigen, cross-reacting with an antigen in Treponema pallidum (Nichols pathogenic strain). Sera from patients with secondary syphilis contain precipitating antibodies against TR-b. The isolation of TR-b from a bacterial sonic extract is described here. It involved five fractionation steps: anion-exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22 Ultrogel), and affinity chromatography on phenyl-Sepharose CL 4B, iminodiacetic acid-Sepharose CL 4B, and lysine-Sepharose 4B, respectively. The purified TR-b was enriched 199 times compared with the starting material, and the recovery was 12%. TR-b was shown to be a protein; it did not bind to a series of lectins, and by gel filtration and polyacrylamide gel electrophoresis, the molecular weight was determined to be 610,000 to 630,000. It was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of identical 70,000-dalton subunits. The isolated TR-b was immunologically pure when tested in crossed immunoelectrophoresis against polyspecific anti-Reiter immunoglobulin. The purified TR-b antigen was used for the production of a monospecific rabbit antiserum, giving strong fluorescence with both the Reiter treponeme and T. pallidum in an indirect immunofluorescence test.

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody against the Reiter treponeme flagellum in syphilis.

An enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies against the periplasmatic flagellum of the Reiter treponeme is described. IgM in the test samples was bound to anti-IgM-coated microtest plates, and flagellum-specific IgM antibody was subsequently detected by incubation with a purified flagellum preparation and monospecific anti-flagellum conjugate. Rheumatoid factor, antinuclear antibodies, or flagellum-specific IgG did not interfere. The specificity of the ELISA for IgM antibodies was 99.5% for sera from 200 blood donors and 98.6% for 147 patient sera that gave false-positive reactions in other syphilis serological tests. The sensitivity was 88.5% for sera from 87 patients with first-time primary syphilis, 93.5% for sera from 62 patients with first-time secondary syphilis, 21.4% for sera from 42 patients who were reinfected, and 0% for sera from 13 patients with late syphilis. Of the sera from 153 patients with treated syphilis, 7.2% had IgM antibodies, and sera from patients with primary or secondary syphilis generally had no IgM antibodies 6 months after treatment. The finding of IgM antibodies indicates that patients should receive antisyphilis treatment if they have not been treated recently, but a negative result does not exclude the possibility of active syphilis. The method may prove useful for the diagnosis of congenital syphilis in newborns.

Isolation and partial characterization of prochymosin and chymosin from cat.

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.

Serodiagnosis of syphilis by an enzyme-linked immunosorbent assay for IgG antibodies against the Reiter treponeme flagellum.

An enzyme-linked immunosorbent assay for IgG antibodies against the flagellum (axial filament) of the Reiter treponeme (flagellum-ELISA) was developed and compared with the fluorescent treponemal antibody absorption (FTA-ABS) test and the Treponema pallidum immobilization (TPI) test with regard to diagnostic sensitivity and specificity. One serum from each of 827 individuals with and without syphilis was studied. In all diagnostic groups of syphilis there was no significant difference between the sensitivity of the FTA-ABS and the flagellum-ELISA, except in treated syphilis, where the FTA-ABS was more sensitive (P less than 0.01). In primary syphilis and in treated syphilis the sensitivity of the flagellum-ELISA was higher than the sensitivity of TPI (P less than 0.01 and P less than or equal to 0.05), respectively); in all other groups there was no significant difference between the sensitivity of TPI and flagellum-ELISA. The specificity of the flagellum-ELISA (99.0%) in 200 sera from blood donors without syphilis was not statistically different from the specificity of FTA-ABS (98.0%) and TPI (99.5%). The flagellum-ELISA seems to be well suited for routine serodiagnosis of syphilis and may replace other treponemal tests.

Purification of a Reiter treponemal protein antigen that is immunologically related to an antigen in Treponema pallidum.

A protein antigen called TR-o was isolated from supernatant of a sonically treated Reiter treponeme. The isolation procedure included anion-exchange chromatography on Whatman DE-52, hydrophobic interaction chromatography on decyl agarose, and finally gel filtration on Ac-A-22 Ultrogel. The fractionations were monitored by immunoprecipitation techniques. The recovery was found to be 35%, and the isolated protein was enriched 220 times. The molecular weight of the native protein was estimated to be 550,000 by polyacrylamide gel electrophoresis and 450,000 by gel filtration. Only one 66,000-molecular-weight polypeptide was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. The protein was immunologically pure when tested in crossed immunoelectrophoresis against polyspecific rabbit anti-Reiter immunoglobulin, detecting more than 40 treponemal antigens. A monospecific antiserum was raised in rabbits immunized with the purified protein. Monospecific rabbit anti-TR-o gave strong fluorescence with both the Reiter treponeme and Treponema pallidum. The corresponding antigen in T. pallidum could not be demonstrated directly in a crude T. pallidum sonic extract, but rabbit anti-T. pallidum immunoglobulin contained precipitating antibodies against the purified protein. No antibodies against TR-o were found in selected sera from patients with secondary syphilis reactive in traditional syphilis tests.

Purification of the synaptic membrane glycoprotein D2 from rat brain.

D2 is a nervous-specific membrane protein enriched in fractions of synaptosomal membranes from rat brain. Recently, an immunochemical relationship between D2 and the chick cell adhesion molecule (CAM) has been demonstrated. There is reason to believe that D2 is involved in adhesion phenomena between neurites. The purpose of the present study was to purify and further characterize the D2 protein from rat brain. In the developed purification procedure synaptosomal membranes from rat brains were prepared and solubilized by means of non-ionic detergent. The subsequent purification steps were hydroxylapatite chromatography, wheat germ lectin affinity chromatography, gel filtration, and lysine affinity chromatography. The purified D2 was found to be enriched 240 times compared with the starting brain homogenate and 120 times compared with the synaptosomal membrane fraction. The recovery of D2 was 26% when the amount of D2 in the synaptosomal membrane fraction was set to 100%. The purified D2 antigen was used for production of monospecific rabbit antisera, and it was found to be composed of two polypeptides of apparent molecular weights 130,000 and 150,000, respectively.