In principle, the test is performed in a similar manner to other well
known slide tests. A certain amount of concentrated killed antigen and
patients serum are mixed on a plate, slide or card and allowed to react
for a specified period, after which the presence of agglutination is determined
by naked eye.
Slide-test antigens are usually suspensions of leptospires that have
been killed by formalin or thiomersal.
Serotypes can be mixed into pools without loss in sensitivity.
Example of pools of leptospira antigen:
||Hond Utrecht IV
||Control formaline-glycerol solution.
Antigens which show clumps or auto-agglutination should be discarded.
The antigens are usually stable for at least one year, but should be tested
for stability and sensitivity before use. For rapid screening, the batteries
of 12-21 antigens are combined into 4-7 pools, each with 2/3 antigens and
representing one serogroup. One or more of these pools will react with
most antibodies induced by the infecting strains of leptospires. Locally
prevalent strains should be included. Reactive sera should be tested by
MAT to determine the antibody titer and the presumptive serogroup of the
PREPARATION OF ANTIGENS FOR THE MACROSCOPIC SLIDE TEST
Leptospires are grown in EMJH medium. Tubes with 3 ml medium are inoculated
with a few drops of a 5-7 day old leptospira culture. The inoculated tubes
are incubated at 30°C for 5-7 days. After inoculation the cultures
are examined by dark-field microscopy. If they are satisfactory (abundant
growth and free of contamination) the cultures are dispensed into an Erlenmeyer
flask containing 50 ml EMJH medium and are then incubated for 5-7 days
at 30°C in a shaking incubator. After incubation and microscopic examination,
the cultures are dispensed in 500 ml of EMJH medium into a 1 liter flask,
and incubated again for 5-7 days at 30°C in a shaking incubator. After
this, the cultures are examined again y dark-field microscopy. If they
are satisfactory, formalin is added to a final concentration of 0.5% and
the cultures are allowed to stand for at least 30 minutes.
The cultures are centrifuged for 30 minutes at 10,000 rpm to pack the leptospires.
A rotation speed of less than 10,000 can also be used (for instance 4,000
- 5,000 r.p.m. for 1 hour or more).
The supernatant is discarded carefully and the tubes are allowed to drain
in a slanted position for 2 hours.
The sediments in each tube are resuspended in 1.5 ml of a solution containing
0.5% formalin, 12% NaCl and 20% glycerol in distilled water, added carefully.
The addition of glycerol to the slide antigens prevents rapid drying during
the test and has been found also to protect the stability of the antigens.
Hyper tonic NaCl is selected because it has a surface tension which prevents
antigen-serum mixtures from spreading too much during rotation of the glass
Pool the suspension in a 100 ml flask. Add glass beads and shake vigorously.
The antigens are kept at 4°C for 2 weeks, before standardization.
If after 2 weeks a deposit has formed, pipette the supernatant into another
bottle and discard the deposit.
Measure the optical density at 520 nm in a spectrophotometer
with quartz cuvettes. Adjust to a reading of 0.550-0.600 by diluting the
suspension with the NaCl-formalin-glycerol solution.
The slide test antigens have to be tested in the macroscopic slide test
as well as in the microscopic agglutinations test. Titers should be 1:320
MICROSCOPIC TEST FOR THE CONTROL OF THE SLIDE TEST ANTIGEN
Make doubling dilution starting at 1:10 of the homologous antiserum in
a microtitre plate in PBS pH 7.2
Transfer 10 ul of the serum dilution onto a glass plate.
Add 50 ul of the antigen to each droplet of diluted antiserum. The first
dilution is now 1:60
Mix diluted serum and antigen with an applicator stick. Avoid air-bubbles.
Place the plate on the mechanical rotator for minutes (125 rpm) or by hand.
Remove the plate and read the reaction over an indirect light source.
The degree of agglutination is recorded as 4+ when all organisms appear
clumped, 3+ if appr. 75% have agglutinated, 2+ with 50% agglutination and
1+ with 25%. No agglutination, characterized by an even suspension of serum-antigen
mixture, is recorded as negative.
MICROSCOPIC AGGL. TEST FOR THE CONTROL OF SLIDE TEST ANTIGEN
Make doubling dilution starting at 1:10 of the homologous rabbit antiserum
in a microtitre plate (10 ul of rabbit antiserum + 90 ul PBS).
And 50 ul of slide test antigen to 50 ul of serum dilution. The first dilution
is now 1:20. Use live and killed (0.5% formalin final concentration) leptospires
Incubate at 30°C for 2-4 hours.
Examine the serum-antigen mixtures by dark-field microscopy for agglutination,
after transferring one drop of the mixture to a microscope slide. The end-point
titer is that dilution which gives 50% agglutination, leaving 50% free
bacteria, compared with a control suspension of leptospires diluted 1:2
in PBS without serum.