Sero-diagnosis of gonorrhoea with a microprecipitin test using a lipopolysaccharide antigen from N. gonorrhoeae.

Abstract

Serum tests for gonorrhoea, particularly the complement-fixation procedure using whole gonococcal cells as the antigen, have long been recognized to be of very limited value in diagnosis. For more efficient diagnosis and control of gonorrhoea there is clearly a need for a simple serological method which can be used as a screening test aimed particularly at the detection of the female carrier in circumstances in which discretion is required and in which routine genital examination is not practicable.In this context a simple "microprecipitin ring test", using a specific lipopolysaccharide antigen of gonococcal origin, has been devised. Preliminary trials have shown its possible usefulness (Chacko and Nair 1966a). The present report gives the results of a more extensive evaluation. The kinetics of the precipitin ring test were described by Nair and Chacko (1966).Material and Methods Preparation of the Antigen The specific antigen was prepared as follows:A 24-hour growth of a virulent strain of gonococcus (F62-type i, obtained through the courtesy of the VDRL, Atlanta, U.S.A.) grown in Chacko-Nair selective medium (I966b, i968) was emulsified in broth and o01 ml. aliquots were inoculated into the allantoic cavity of 8-day-old chick embryos through the air sac. Eggs were candled periodically and at the end of 48 hours' infection and growth of gonococci, they were kept in the refrigerator at 40C for 2 to 4 hours. The allantoic fluid was then aspirated under sterile conditions using a io-ml. syringe and an i8-gauge needle and the purity of the cultures was checked by Gram-staining and sub-culture.Next, the allantoic fluid was centrifuged lightly to remove all the macroscopic and fat particles. It was then pipetted into large centrifuge bottles and six *Received for publication September 12, 1967 tProfessor of Serology tAssistant Serologist volumes of absolute alcohol were added; the mixture was shaken thoroughly and kept at 5 to io0c for 24 to 48 hours. The contents were then centrifuged at 2,000 r.p.m. for one hour, the supernatant was discarded, and any excess alcohol burnt off. Half the original volume of double-distilled deionized water was added to the precipitate, which was then homogenized by using a 5-mi. pipette, was again centrifuged at 2,000 r.p.m. for one hour, and the supernatant watery extract pipetted out. An equal quantity of absolute alcohol was added and centrifuged at 2,000 r.p.m. for 30 minutes. To the supernatant, 6 volumes of absolute alcohol were again added, followed by a trace of sodium acetate and 4 to 8 ml. glacial acetic acid, the whole was mixed well and kept in the cold for 2 hours. The contents were centrifuged at 2,000 r.p.m. for 45 minutes and the deposit was dissolved in a volume of water one-eighth of the original volume of culture fluid taken. After further centrifugation to remove all the insoluble particles, the slightly turbid supernatant so obtained was titrated to obtain the exact units of antigen necessary for the test, as follows: Doubling dilutions of the original antigen pre...

Evaluation of the gonococcal complement-fixation test.

“…A microprecipitin test (Chacko and Nair, 1969) shows promise, but still cannot differentiate between past and present gonorrhoea. Until the superiority of these tests is established it would be unwise to discard the GCFT altogether.…”

Quantitative Determination of Antibody to Gonococcal Pili. CHANGES IN ANTIBODY LEVELS WITH GONOCOCCAL INFECTION

“…Of the 103 females, those with negative cultures at all sites had a lower geometric mean antibody activity (1.1 Ag/ml) than those with positive cultures (4.2 ,g/ml, P <0.001). 50 (86%) of the 58 females with more than 1.6 Ag/ml antibody activity had cultures which grew gonococci, as compared to eight (18%) of 45 for those with 1.6 /ug/ml or less. Six of the eight asymptomatically infected females whose antibody activity was less than 1.6 iFg/ml were women examined in a venereal disease clinic and two were girls in a reform school.…”

“…8). Culture results negative (45) positive (58 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). The use of a pure antigen preparation decreases the chance of detecting crossreacting antibody responses to organisms other than gonococci, and is a prerequisite to quantitating the antibody response to a single antigen.…”

Human antibody response to lipopolysaccharides fromNeisseria gonorrhoeae

“…A very sensitive microprecipitin test described by Chacko and Nair (1969) detected antibody in 60% of patients with a urethral discharge of less than three days' duration. The antigen was extracted with ethanol from the allantoic fluid of hens' eggs which had been infected with gonococci, but although the authors suggest it was a lipopolysaccharide, it must have been degraded as it was unable to produce a Schwartzman reaction.…”