Practical No.

: 5
Title: DIAGNOSIS OF LEPTOSPIROSIS

1. Microscopic Agglutination Test For The Detection Of Antibodies To Leptospires

Principle: Antibodies in the test serum react with antigens on the surface of the bacteria and
agglutinate them.
Materials: Microtitre plates; Incubator 30C; Loop; Microscope slides; Microscope (equipped
with objective 29x, dark field condenser and 10x eyepieces); Microtitre pipettes.

Reagents:
Leptospira cultures; PBS, pH7.2
Procedure:
1. Fill all 96 wells of a microtitre plate with 50 uL PBS.
2. Add another 40 uL PBS to the wells of column 2.
3. Add 10 uL of antiserum to the wells of column 2 (serum dilution is now 1:10).
4. Dilute by pipetting 50 uL from one well to the next. Discard the final 50 uL.
5. Add 50 uL Leptospira culture to all wells (dilution in wells from column 2 is now
1:20) and cover the microtitre plate.
6. Mix thoroughly on a micro shaker for a few seconds.
7. Incubate for 2-4 hours at 30C or overnight at room temperature.

Reading the results:

The serum-antigen mixtures are examined under a dark field microscope for agglutination.
This can be done by transferring one drop of mixture to a microscope slide.
The endpoint (titer) is taken as that dilution which gives 50% agglutination, leaving 50% of
the cells free. Compare with a control suspension of leptospires diluted 1:2 in PBS without
serum (column 1). N.B. Agglutination is not always visible under the microscope.

2. ELISA Test For The Detection Of Antibodies To Leptospires

PRODUCT DISCRIPTION
ELISA was used to detect specific IgM and IgG in sera from humans with current or past
leptospirosis. A serological pattern of a high IgM titre, or moderately increased IgM in
conjunction with a low IgG titre, with serovar copenhageni antigen was characteristic for
approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in
the first two months of the disease. The antigen was the supernatant of a heated and
centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni,
grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa
and hardjo infections showed cross-reactivity when different antigens were used. In past
infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM
and IgG allows the rapid diagnosis of acute leptospirosis.

2. SIZE OF THE PACK

96 well plate -5
8well plate-5(12 strips)

3. PACKING LIST
 Leptospira Microplate (IgM/gG)
 Sample Diluents
 Stop Solution
  Washing Solution (20x conc.)
 Leptospira Solution 1
 Leptospira Solution 2
 Streptavidin conjugate
 TMB Substrate Solution
 Leptospira IgM/IgG Positive Control
 Leptospira IgM/IgG Cut-off Control
 Leptospira IgM/ IgG Negative Control
 Strip holder
 Cover foils

Materials Required But Not Provided:

 Distilled or deionized water
 Precision pipettes: 5 μl, 10 μl, 50 μl, 100 μl and 1.0 ml
 Disposable pipette tips
 Microtiter well reader capable of reading absorbance at 450/620 nm.
 Vortex mixer, or equivalent
 Incubator 37°C
 Manual or automatic equipment for rinsing wells
 Disposable tubes
 Timer

PROCEDURE: