RAPID LATEX AGGLUTINATION ASSAY (LEPTO RLA)

INTENDED USE

The Lepto rapid Latex agglutination assay has been developed for the serodiagnosis of human leptospirosis. It is designed for the detection of Leptospira-specific agglutinating antibodies in human serum samples.

INTRODUCTION

The human Lepto RLA is a simple assay for the detection of Leptospira-specific agglutinating antibodies in human sera. This assay is rapid, and requires no specialized equipment. The ingredients are highly stable. After opening of the vials the reagents should be stored at + 4 C. When tested on a group of sera from leptospirosis patients and negative controls from The Netherlands the performance of the assay was found to be similar to that of the MAT performed on a panel of 14 different strains.

PRINCIPLE

In the Lepto RLA Leptospira-specific agglutinating antibodies in serum samples are detected by the binding of the specific antibodies to blue latex particles coated with Leptospira specific antigen. Binding of antibodies becomes visible by the formation of colored aggregates when a serum sample is mixed with a suspension of latex particles .The assay is performed by mixing on a white agglutination card 5 ul of a serum sample and 5 ul of the latex. The assay is read within 2 minutes after mixing. For an optimal result the agglutination card is swirled continuously for the duration of the assay. A reaction with control latex particles containing no antigen is performed in parallel to check for non-specific reactions. The use of a broadly reactive antigen of a pathogenic leptospira for coating of the latex particles ensures the efficient detection of a wide spectrum of Leptospira infections.

REAGENTS

• 200 ul lyophilized test latex (Vial A)
• 200 ul lyophilized control latex (Vial B)
• 500 ul reconstitution fluid (Vial C).

Warning: The latex reconstitution fluid contains sodium azide. Do not swallow and avoid skin contact.

• Lyophilized positive control serum (Vial D)
• Lyophilized negative control serum (Vial E)
• Five agglutination cards for the performance of 4 assays each.

UTENSILS (NOT PROVIDED)

• 100 ul micropipete
• 5 ul micropipete
• Disposable micropipete tips.
• Stopwatch

PACKAGE SIZE

Each package contains sufficient reagents for the analysis of 19 sera including the positive and negative control sera.

STORAGE

Reagents should be stored at +4 C to 37 C protected from direct exposure to sunlight. After reconstitution reagents should be used within four months. Reconstituted reagents should be stored at +4 C.

EXPIRY DATE

The ingredients expire on the date printed on the box.

PRECAUTIONS

Blood and serum samples should be handled carefully as they may contain infections agents. Equipment and supplies for specimen handling should be treated with due caution. Serum samples which have been heat-inactivated (56 C, 30 minutes) may be used as heat treatment does not affect the result of the assay. Used disposables and samples should be properly discarded.

SPECIMEN COLLECTION

Serum should be collected in the same way as routinely performed for any clinical laboratory assay.

STANDARD ASSAY PROCEDURE

Open Vial A containing the lyophilized test latex and Vial B containing the lyophilized control latex. Open Vial C containing the reconstitution fluid and transfer 100 ul to Vial A. Reconstitute the test latex by pipetting up and down five times. Using a new pipette add 100 ul reconstitution fluid (Vial C) to vial B and reconstitute the control latex by pipetting up and down five times.

Mark an agglutination card with the identification number of the serum to be tested (one card provides sufficient place for testing four sera, but only one serum should be tested at one time). Using a micropipet and disposable tips place two drops of 5 ul serum on the agglutination card below the serum identification number (one drop in the row marked C for control and one drop in the row marked T for test). Add 5 ul control latex suspension (Vial B) to the drop of serum placed in row C. Mix serum and control latex suspension using the tip of the piper.

Using a new tip, immediately proceed by adding 5 ul test latex suspension (Vial A) to the drop of serum placed in row T again mixing the serum and test latex suspension using the tip of the pipette.

Continue the procedure by mixing latex suspension and serum by hand rotation of the card for a maximum of 2 minutes during which time period aggregates may be formed with the test latex revealing the presence of Leptospira specific antibodies. If no aggregates are formed within 2 minutes the sera may be considered negative. aggregation occurring after 2 minutes may be due to evaporation of the liquid.

Read the result of the assay during rotation of the card. In case of a strong reaction aggregates become visible immediately after the addition of the test latex. In case of a weak reaction aggregate formation may take up to 2 minutes.

INTERPRETATION OF ASSAY RESULTS

Formation of aggregates with the test latex shows the presence of Leptospira-specific IgM antibodies in the serum sample. If aggregate formation is weak, i.e. occurring after 1.5 to 2 minutes only or not present, and suspicion of leptospirosis remains, the assay should be performed on a second sample taken at a later to look for a stronger reaction or seroconversion. If the control latex reveals aggregates, the assay is invalid and the sera should be analyzed with another assay.

The presence of antibodies is evidence of current, recent or past exposure to leptospires.

LIMITATIONS OF USE

As with any diagnostic test, results from the Lepto RLA should be interpreted with consideration of the clinical and other laboratory findings.

Antibodies usually become detectable 7-10 days after the onset of the disease and sometimes even later. Early antibiotic treatment may suppress antibody formation.

Although the results of the Lepto RLA are found to be in agreement with those of the MAT for the detection of Leptospira-specific agglutinating antibodies, neither 100% sensitivity nor 100% specificity is claimed.

False-positive reactions may occasionally occur with sera from patients with diseases other than leptospirosis. However, only a weak degree of agglutination has been observed in such cases.

False-negative reactions occasionally may occur when either only serovar specific antibodies are formed.

In case of doubt, confirmation with MAT, ELISA and/or Dipstick assay is recommended.

REFERENCES

Hoorn, M.A.W.G. van der Goris, M.G.A. Korver, H. Mesa-Brewster, J.de Ingen, C.van Terpstra, W.J. Harktskeerl, R.A. and Smits, H. L. Evaluation of a Latex Agglutination Assay for the quick serodiagnosis of leptospirosis in humans. In preparation.

Faine, S. Guidelines for the control of leptospirosis. World Health Organization, Geneva, 1982.