Author: Miladin Kostovic

## Standardization of typing methods

Standardization of methods is a self-evident necessity in classification studies in order that comparable results may be obtained by the different laboratories. During each meeting of the TSC this item has been discussed but some questions still remain unanswered. However a number of recommendations were agreed at the TSC meetings in Munich 1978 (Minutes, 1982) and Boston 1982 (Minutes, 1984).

#### The preparation of rabbit immune serum.

For classification purposes, a pooled antiserum from two or three rabbits should be used with a titre between 10.000 and 50.000. It is recommended to prepare the sera by repeated intravenous injections of well grown living culture of an approximate density of 2 x 10(8) organisms per ml. (see for more details Minutes 1982 of TSC meeting Minich, 1978). One must take into account the possibility that in some strains there may be a thermolabile antigen(s) as well as the usual thermostable antiens (Borg-Peterson, 1971, 1972; Babudieri, 1972; Kmety, 1972). In order to distinguish both types of antigen other methods of immunization have been adopted (Dikken and Kmety, 1978).

#### Microscopic agglutination test (MAT).

In standarizing the MAT, two important points should be noted:

a. the correct density of the culture used as antigen;

b. the determination of the end-point of agglutination (titre).

a. The density of the antigen is known to influence the end-point of agglutination and consequently the titre of the serum (Borg-Petersen and Fagreus, 1949; Jarekova, 1986). therefore the TSC (WHO, 1965; Minutes 1984 of TSC meeting Boston, 1982) recomminded the use of well-grown living cultures with an approximate density of 2 x 10(8) leptospires per ml. However, differences in the length of the leptospiral cells may influence the real density of the reacting antigen determined nephelometrically (Jerekova, 1986) who found lower densities (approximately 1,5 x 10(8) leptospires per ml) to be more suitable for the test.

b. The end-point of the reaction of defined as the reciprocal of the highest dilution of serum in the serum-antigen mixture, in which 50% or just more of the cells are agglutinated (WHO, 1965; Minutes, 1984 of TSC meeting Boston, 1982).

#### Agglutinin-absorption test.

The absorption test should be well balanced in order to ensure that neither over nor under absorption take place. To achieve this, the amount of concentrated living absorbing antigen with a density corresponding to McAarland standard No. 10 (Dikken and Kmety, 1978) or determined otherwise (Minutes, 1984 of TSC meeting Boston, 1982) should be appropriate to the height of the antibodies of the immune serum to be absorbed. If lower titre antibodies, are to be absorbed, lower amounts of absorbing antigen have to be used to achieve a well balanced absorption.

It is recommanded that 1 part of immmune serum should be mixed with 24 parts of concentrated antigen in 3 equal amounts at 10 minute intervals. The absorption is considered to be adequately balnced when the absorbed serum has a residual titre of 0,5 - 1,0% of its original titre against the absorbing antigen (see for more details Minutes, 1984 of TSC meeting Boston, 1982). Even in the case of complete absorption the test may be considered in balance provided nosubstantial reduction of the homologous serum titre is apparent.