Detection of Clostridium difficile Toxin A by Reversed Passive Latex Agglutination

Abstract

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).

Detection of Biological Agents: Looking for Bugs in All the Wrong Places

“…19,20 Imm unoassays such as ELISA and assays using PCR are also employed. [21][22][23] Spectroscopic Methods for Detection and Identi cation. To address the need for rapid and sensitive pathogen detection, particularly the detection of BA, there is a need for rapid, compact, eld-portable, userfriendly equipm ent.…”

“…37 Naumann and co-workers divided the entire infrared spectrum into ve main regions based on their discriminative power and speci c information. These regions are as follows: the fatty acid region (3000 -2800 cm 2 1 ), which is dominated by the -CH 3 , the .CH 2 , and the 5CH stretching vibrations of functional groups typically present in the cell membrane; the amide region (1800 -1500 cm 2 1 ), which is dominated by amide I and amide II stretchings of peptides and proteins; the m ixed region (1500 -1300 cm 2 1 ), where proteins and fatty acids are seen; the phosphate and polysaccharide region (1300 -900 cm 21 ), which is dominated by carbohydrates in the cell wall an d com p oun ds con tain ing p ho sphates; and the true ngerprint region (900 -700 cm 2 1 ), which shows very speci c patterns that have not yet been assigned to speci c functional groups. [33][34][35][36] Species and strain identi cation can be accomplished by performing a cluster analysis on the rst derivatives of a selected wavelength region or the entire spectrum.…”

National Clostridium difficile Standards Group: Report to the Department of Health

“…Toxin production can be demonstrated by putting broth culture filtrates through the same EIAs used for faecal specimens, and also by the cell cytotoxicity assay. 105 Alternative methods reported include reversed passive latex agglutination (applied to toxin A), 106 colony blot probe-hybridisation (applied to toxin B), 107 and PCR (toxins A and B). 108 These so-called 'second-look' cytotoxicity assays can be shown to detect toxigenic C. difficile in diarrhoeal specimens negative in the stool cytotoxicity assay, but the clinical significance of this finding requires confirmation.…”

Taxonomic position of new bacteriocin (nukacin ISK-1) producer isolated from long-aged Nukadoko.

“…Enterotoxin was detected by TST-RPLA "SEIKEN," a reagent kit for detecting Staphylococcus TSST-1 (Toxic Shock Syndrome Toxin) and SET-RPLA "SEIKEN" Staphylococcal enterotoxin A, B, C, and D detection kit by reversed passive latex agglutination (Denka Seiken Co., Ltd., Tokyo, Japan) (Toma et al, 1999). None of the enterotoxins were detected.…”