Group-Specific Hemagglutination Test For Neisseria meningitidis Antibodies

Abstract

The significance of human carriers of Neisseria meningitidis and meningococcal disease is obscured by the lack of an easily performed and specific serological test (1). None of the serological methods so far described for the serodiagnosis of meningococcal carrier status or disease has proven entirely satisfactory.

In the past several years, indirect or passive hemagglutination (PHA) has become one of the most widely used methods for measuring various antibodies. A number of techniques has been devised for the attachment of antigen to the erythrocyte (rbc) surface( 2 3 4 5 6 7 8 9 ). The present report describes a modification of 1 of these( 8 ) that resulted in an antigen from N. meningitidis which would absorb to a sheep rbc. This rendered the sheep rbc agglutinable in the presence of antiserum homologous to the sensitizing antigen. Its use in the PHA test for the measurement of N. meningitidis antibodies is also described.

Methods. Antigen preparation. Stock cultures of N. meningitidis groups A (Cl-4),∗ B (16B6),∗ C (PTS-5),∗ Bo,† and 29E† were used to prepare antigens. One ml of an 8-hour Mueller-Hinton‡(10) broth shake culture of each group was inoculated into 250 ml Mueller-Hinton broth and incubated for 18 hours at 37°C in a Psycrotherm Incubator shaker§ (170-200 rotations/minute) with normal atmospheric air.

As a safety precaution, cultures were then inactivated with beta-propiolactone (1% final concentration) for 1 hour at room temperature and overnight at 6°C. The sediment was collected by centrifugation (600 × g), washed 3 times with 0.15 M NaCl (saline) and resuspended in 101 ml of saline. The suspension was adjusted to pH 11.0 with 1 N NaOH, incubated for 1 hour at room temperature and then readjusted to pH 6.5 with 1 N HCI. The suspension was precipitated with 5 volumes of absolute ethanol. The precipitate was collected by centrifugation (3000 × g) and resuspended in 20 ml saline. The insolulble residue was removed by centrifugation and discarded.