Slide agglutination test

MACROSCOPIC SLIDE TEST

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:

Pool number | Reference strain | Serovar
Pool 1 | M 20 RGA | copenhageni icterohaemorrghagiae
Pool 2 | Hond Utrecht IV Mus 127 | canicola ballum
Pool 3 | Moskva V Pomona | grippotyphosa pomona
Pool 4 | Jez Bratislava Ballico | bratislava australis
Pool 5 | Hebdomadis Mus 24 Sari | hebdomadis saxkoebing mini
Pool 6 | Poi Perepelicin van Tienen | poi tarassovi bataviae
Pool 7 | Patoc I | patoc
Pool 8 | 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 infecting strain.

PREPARATION OF ANTIGENS FOR THE MACROSCOPIC SLIDE TEST

1. 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.
2. 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).
3. The supernatant is discarded carefully and the tubes are allowed to drain in a slanted position for 2 hours.
4. 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 test plate.
5. Pool the suspension in a 100 ml flask. Add glass beads and shake vigorously.
6. The antigens are kept at 4°C for 2 weeks, before standardization.
7. If after 2 weeks a deposit has formed, pipette the supernatant into another bottle and discard the deposit.
8. 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.

STANDARDIZATION

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 or higher.

MICROSCOPIC TEST FOR THE CONTROL OF THE SLIDE TEST ANTIGEN

1. Make doubling dilution starting at 1:10 of the homologous antiserum in a microtitre plate in PBS pH 7.2
2. Transfer 10 ul of the serum dilution onto a glass plate.
3. Add 50 ul of the antigen to each droplet of diluted antiserum. The first dilution is now 1:60
4. Mix diluted serum and antigen with an applicator stick. Avoid air-bubbles.
5. Place the plate on the mechanical rotator for minutes (125 rpm) or by hand.
6. Remove the plate and read the reaction over an indirect light source.
7. 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

1. Make doubling dilution starting at 1:10 of the homologous rabbit antiserum in a microtitre plate (10 ul of rabbit antiserum + 90 ul PBS).
2. 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 as controls.
3. Mix thoroughly.
4. Incubate at 30°C for 2-4 hours.
5. 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.