Stabilized, freeze-dried PCR mix for detection of mycobacteria.

We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4 degrees C and at 20 degrees C for 1 year and for 3 months at 37 degrees C, as judged by their performance with 50 and 500 fg of purified Mycobacterium bovis BCG target DNA.

A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae.

Among the many reported applications of the detection of antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae, in particular, the use of seroprevalence as an indicator of the magnitude of the leprosy problem may turn out to be very useful in leprosy control programs. An operational function of serology within the leprosy control services requires a simple test system. We have developed a simple dipstick assay for the detection of antibodies to PGL-I and compared its performance with that of an ELISA. A high degree of agreement (97.2%) was observed between the ELISA and the dipstick assay when tested on 435 sera; the agreement beyond chance (Kappa value) was 0.92. No significant difference was found between the dipstick assay and the ELISA when seropositivity rates obtained in groups of leprosy patients, household contacts, and controls were compared. The interpretation of the dipstick results as positive or negative was unequivocal, as illustrated by the high agreement between different persons reading the test (Kappa values > 0.88). Storage of the only reagents required, the dipsticks and the stabilized detection reagent, up to three weeks under tropical conditions of high temperatures, high humidity, and exposure to light, did not influence the results of the assay. The dipstick assay described here is an easy-to-perform method for the detection of IgM antibodies to PGL-I of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries. An internal control validates the performance of the assay. This dipstick assay may be the method of choice for epidemiologic mapping of leprosy.

LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera.

We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis.

We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti-Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.

Leish-KIT, a stable direct agglutination test based on freeze-dried antigen for serodiagnosis of visceral leishmaniasis.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.

The influence of sucrose, dextran, and hydroxypropyl-beta-cyclodextrin as lyoprotectants for a freeze-dried mouse IgG2a monoclonal antibody (MN12).

The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-beta-cyclodextrin (HP beta CD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56 degrees C). For comparison, solutions of MN12 were stored under the same conditions. Regardless of the lyoprotectant used, precipitation and a concomitant reduction of the antigen-binding capacity by about 10% were observed upon reconstitution of lyophilized MN12. HP beta CD proved to be the most effective stabilizer to prevent degradation of lyophilized MN12 during storage. Compared with MN12 solutions, HP beta CD-containing lyophilized MN12 cakes were more resistant to heat-induced charge alterations and loss of antigen-binding capacity.