Coagglutination Research Articles

Surface immunolabeling of Neisseria gonorrhoeae employing monoclonal antibodies to proteins IA and IB

The technique of immunomarking intact bacteria with monoclonal antibodies (MAb) and gold-labeled anti-IgG provides a rapid and reliable method for localization of surface-exposed antigens. Cell suspensions of Neisseria gonorrhoeae were incubated with monoclonal antibodies directed against different epitopes of protein I as shown by ELISA inhibition test (unpublished data). The bound IgG molecules were detected with gold labeled anti-mouse IgG or protein A. MAbs against various epitopes of protein I showed differences in their reaction pattern with intact bacteria. We compared the results achieved by the gold immunomarking technique with coagglutination (CoA) and immunofluorescence (IF) tests. The results of the three methods correlated for some antibodies and were ambiguous for other MAbs. The advantage of the immunogold method over immunofluorescence and coagglutination is that distribution and localization of antigen on the surface of gonococci can be exactly determined using electron microscopy.

Rapid diagnosis of respiratory infection due to Streptococcus pneumoniae

Microscopic examination of gram-stainedsmear and antigen detection by coagglutination (COA) using sputum specimens obtained from patients with respiratory infection were carried out as a rapid diagnosis of pneumococcal infection. Of the 180 gram smear positive specimens, 84 were positive for COA.When microscopically purulent sputum containing more than 25 leukocytes per low power field and less than 25 epithelial cells per low power field were cultured quantitatively, S. pneumoniae were recovered at a frequency of 88.1% from COA positive specimens in numbers of 105 cfu/ml or more.In contrast the frequency was only 48% using the specimens heavily contaminated with oropharyngeal flora. Most COA positive reactions observed with such specimens seemed to be falsepositive due to contaminated a-hemolytic streptococcus other than S. pneumoniae.Moreover, in purulent sputum, 65 out of 89 culture positive specimens (73%) were predictable by microscopic examination of sputum smears, while the predictability was 48.3% in contaminated samples.These results clearly showed that Gram-smear and COA are reliable techniques for true purulent sputum in establishing a presumptive diagnosis of pneumococcal infection.

Heterogeneity ofCampylobacter pylorias Demonstrated by Co-agglutination Testing with Rabbit Antibodies

The indirect immunofluorescence (IFL) and the co-agglutination (CoA) methods were used to study the serology of Campylobacter pylori strains isolated from patients in different countries (Sweden, Finland, Canada and Australia). Antisera were obtained from rabbits immunized with whole cell antigens. With IFL tests the highest serum titers were obtained with C. pylori strains and their homologous antisera. These tests also showed that all the tested strains contained cross-reactive antigens. With the use of the CoA technique strain or type specific heat labile and heat stable antigens were demonstrated, and a provisional "seropattern" of a particular strain could be defined with selected CoA reagents. With the use of such reagents we were able to show that a patient may be infected with multiple C. pylori strains with different sets of surface antigens. The clinical and epidemiological implications of this serological heterogeneity of C. pylori are discussed.

Comparison of serological techniques for the identification of Renibacterium salmoninarum

Summary The optimum conditions for staphylococcal co-agglutination (CoA), latex agglutination (LA) and the fluorescent antibody test (FAT) for the identification of Renibacterium salmoninarum isolated in culture were determined. Their sensitivities were then compared with that of the enzyme-linked immunosorbent assay (ELISA). The ELISA was the most sensitive, followed by CoA, with the FAT and LA jointly the least sensitive. However, all four techniques were sufficiently sensitive for routine identification of R. salmoninarum. The agglutination techniques (CoA and LA) were preferred because of their speed, ease of use and low cost.

Comparison of a coagglutination test with the G M1-ELISA and the Y-1 adrenal cell assay for the detection of heat-labile enterotoxin from Escherichia coli

A number of different assay methods has been developed for the detection of the heat-labile enterotoxin (LT) of ETEC strains isolated from humans and animals. In the present study 40 Escherichia coli strains were subject of a comparative study of the staphylococcal coagglutination (Coa-LT) test, the GM1-ELISA and the Y-1 adrenal cell test. All but two of 20 "LT-ST" or "LT-only" producing ETEC strains gave identical results in both the Coa-LT test and the bioassay. None of the 14 "ST only" producing ETEC strains gave false positive results. It is concluded that the Coa-LT test is a simple and rapid assay method for the routine diagnosis of LT producing ETEC strains.

URINE COUNTERCURRENT IMMUN0ELECTROPH0RESIS FOR THE DIAGNOSIS OF PNEUMOCOCCAL PNEUMONIA

We report the results of a prospective trial in which counterimmunoelectrophoresis (CIE) and coagglutination (COA) have been used for the detection of specific pneumococcal capsular antigens in urine of 215 community-acquired pneumonia. Urines were studied unconcentrated and concentrated by ethanol precipitation and by anultrafilter system (Minicon B 15 concentrator Amicon). CIE was positive in 18 patients. Three of them,had untreated urine positive, 14 ethanol precipitated urine and 12 Amicon concentrated urine. COA was positive in none of these 18 CIE positive patients, and blood cultures only in two. In 3 children coexisted other etiology with a positive CIE. CONCLUSION: Althoug COA of urine does not apper to work sufficiently well to warrant its use, CIE can be useful in the diagnosis of pneumococcal pneumonia in children.

Bacterial antigen detection in cerebrospinal fluid of patients with meningitis

This study compared the sensitivity and specificity of four test systems in detecting Haemophilus influenzae type b, Neisseria meningitidis , Streptococcus pneumoniae , and gram-negative organisms in cerebrospinal fluid (CSF), versus culture. The tests used on CSF from 155 patients with meningitis were the Phadebact coagglutination (CoA) test, the Directigen latex agglutination (LA) test, counterimmunoelectrophoresis (CIE), and the Limulus amebocyte lysate (LAL) test. The sensitivity for patients with bacterial meningitis was 78% ( 18 23 ) for LA, 78% ( 25 32 ) for CoA, and 67% ( 18 27 ) for CIE for detection of H. influenzae type b; 71% ( 10 14 ) for CoA, 100% ( 6 6 ) for LA, and 50% ( 6 13 ) for CIE in detecting S. pneumoniae ; and 33% ( 1 3 ) for LA and 50% ( 2 4 ) for CIE in detecting N. meningitidis . LAL had a sensitivity of 77% ( 37 48 ) in detecting CSF gram-negative endotoxin. The specificities of those with bacterial meningitis for H. influenzae , S. pneumoniae , and N. meningitidis tested by LA were, respectively, 100% ( 35 35 ), 96% ( 50 52 ), and 100% ( 54 54 ); for H. influenzae and S. pneumoniae using CoA 97% ( 62 64 ) and 96% ( 80 83 ); for H. influenzae , S. pneumoniae , and N. meningitidis using CIE 67% ( 18 27 ), 50% ( 6 12 ), and 50% ( 2 4 ). The specificity of LAL was 86% ( 38 44 ). The detection of bacterial antigen from CSF in patients with meningitis by commercial agglutination tests is more sensitive than CIE and is highly specific.

An analysis of Streptococcus pneumoniae identification using biochemical and serological procedures

Five methods for identification of Streptococcus pneumoniae were evaluated with stock strains representing all 83 capsular types and 130 fresh clinical isolates of α-hemolytic stretococci. The identification methods included bile solubility, optochin sensitivity, countercurrent-immunoelectrophoresis (CIEP), and coagglutination (CoA) using laboratory-prepared reagents LPR) and the Phadebact Pneumococcus Test (Phadebact). The Quellung reaction was performed on the 83 capsular types of S. pneumoniae and on the clinical isolates that produced serological cross reactions with the three serological tests and those that were bile-soluble and optochin-sensitive. All 83 pneumococcal types were in complete agreement with each of the different test methods. Of the 130 α-hemolytic streptococci, 26 were identified as S. pneumoniae , and 104 isolates were identified as viridans stretococci using biochemical, physilologic, Quelung, or mouse virulence tests. All 104 viridans stretococci were bile-insoluble and optochin-resistant; however, 63 reacted with either one, both, or all three serological methodsO Pur data suggest that bile solubility and optochin tests were are more reliable for pneumococcal identification than serological methods currently available.

Evaluation of the Directigen™ and Phadebact® Agglutination Tests

Comparison testing of the Directigen latex agglutination (LA) kit, the Phadebact coagglutination (COA) kit, and counter-immunoelectrophoresis (CIE) demonstrated that the commercial LA reagents were slightly more sensitive than the COA reagents for the detection of pneumococcal polysaccharide types 2, 4, 8, 9, 12, 19, 23, 25, 51, and 56, and meningococcal polysaccharide from Group C. The COA reagents were slightly more sensitive than the LA reagents for the detection of pneumococcal polysaccharide type 6A. The sensitivity of LA and COA reagents for the detection of Hemophilus influenzae type b capsular polysaccharide, pneumococcal polysaccharide types 1, 3, 14, and meningococcal Group A were equivalent. Purified meningococcal polysaccharides of Groups B, C, and W135 were detected uniformly by CIE but not with the COA reagent. The COA reagent reacted with antigen of Groups B, C, and W135 from broth culture but with less sensitivity than CIE. In general, CIE was the least sensitive method for detecting bacterial antigens. In addition, the commercial CIE antisera for H. influenzae type b, or meningococcal polysaccharides were susceptible to false-negative results due to antigen excess.

Comparison of coagglutination, latex agglutination, and enzyme-linked immunosorbent assay for detection of Haemophilus influenzae type B infection

Two commercially available kits were compared to an enzyme-linked immunosorbent assay (ELISA) for detection of Haemophilus influenzae type b antigen polyribophosphate in clinical specimens with infections proved by culture. Methods employed by the kits were latex agglutination (LA) and staphylococcal coagglutination (COA). The COA kit detects H. influenzae type b and types a and c-f with a polyvalent antiserum, whereas the ELISA assay and the LA kit detect only type b. A total of 139 specimens (41 spinal fluid, 35 urine, 25 serum, and 38 sputum) were tested. All spinal fluid samples positive by culture were positive by all three procedures. Of urine specimens from patients with a variety of H. influenzae type b infections, 13 of 15 were positive by ELISA, 8 of 15 by COA, and 8 of 14 by LA. Of serum samples collected from the same patients at various times during their illness, 8 of 15 were positive by ELISA, 6 of 15 by COA, and 10 of 15 by LA. Of sputum samples positive by culture for H. influenzae type b, 14 of 17 were positive by ELISA, 9 of 17 by COA, and 4 of 16 by LA. The ability to detect additional serotypes of H. influenzae was shown by the COA kit, which detected H. influenzae type a in spinal fluid from a patient with type a meningitis proved by culture.

Serotyping of Streptococcus pneumoniae strains by coagglutination and counterimmunoelectrophoresis.

A total of 217 Streptococcus pneumoniae strains were serotyped by coagglutination (COA) and counterimmunoelectrophoresis (CIE). With all strains tested, there was full agreement between results obtained by COA and CIE, except for strains belonging to serotypes 7, 14, 33, and 37, which could not be typed by CIE. These strains were serotyped by passive immunodiffusion, results of which were in full agreement with those obtained by COA. Besides having the advantage of identifying strains belonging to all serotypes, COA was also more rapid and economical than CIE.

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.

Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens.

A total of 224 strains were serogrouped by coagglutination (COA) and serotyped by protein I enzyme-linked immunosorbent assay (ELISA). Of these strains, 61 were from patients with disseminated gonococcal infection, 21 were from patients with pelvic inflammatory disease, and 115 were from patients with uncomplicated gonococcal infection in Singapore, the Philippines, and Denmark. Twenty-seven were laboratory reference strains. Of the patient strains, 102 belonged to COA serogroup WI, and all of the 100 strains that typed with protein I serotypes 1, 2, or 3 were in this group. Most of the strains of gonococci from the 61 patients with disseminated gonococcal infection were within this group (COA WI, 53 or 87%; protein I serotypes 1, 2, or 3, 51 or 84%). All 46 strains that were protein I serotypes 4 through 7 were also COA serogroup WII. Protein I serotypes 8 and 9 accounted for 49 (25%) of the 197 patient strains. Twenty-eight of these strains typed as COA serogroup WII, 20 typed as serogroup WIII, and 1 typed as serogroups WII and WIII. COA W serogrouping and protein I ELISA both appeared to detect antigens on the protein I molecule of the outer membrane of Neisseria gonorrhoeae. Protein I serotyping, which uses unboiled organisms, may generally recognize more variable and surface-exposed antigenic determinants. In contrast, COA W serogrouping, which uses boiled organisms, may recognize less exposed shared antigenic determinants in addition to variable protein I antigenic determinants. Both methods may prove useful for further studies of the epidemiology and pathogenesis of gonorrhea.

Evaluation of the coagglutination test for the identification of Neisseria gonorrhoeae in primary cultures.

The coagglutination (COA) test for the identification of Neisseria gonorrhoeae was compared with immunofluorescence and sugar degradation tests on 1710 gonococcal isolates, 72 of which produce beta-lactamase. The COA test gave a positive result for 98.6% of the strains. Treatment of suspensions with Streptomyces enzyme reduced the incidence of inconclusive results due to autoagglutination to 1.2%. Cross-reactivity of the gonococcal antiserum was minimised by absorption with meningococci and Moraxella species. The COA provide a simple, quick, and reliable method for identifying N gonorrhoeae in culture.

SEROLOGY OF CAMPYLOBACTER FETUS SS. JEJUNI (»RELATED« CAMPYLOBACTERS).

Rabbit antisera against two strains of Campylobacter fetus ss. fetus (serotype A), two strains of C. fetus ss. intestinalis (serotypes A and B respectively), and eight stains of C. fetus ss. jejuni were used in serological studies of these strains with the use of co-agglutination (COA), line-rocket immunoelectrophoresis (L-RIE) and rocket-line immunoelectrophoresis (R-LIE). Whole bacterial cells, either heated at 56 degrees C. boiled or atuoclaved, were used in COA tests. Unheated sonicates were used in L-RIE, and sonicates, unheated, boiled or autoclaved, in R-LIE. The antigenic properties of C. fetus ss. fetus and C. fetus ss. intestinalis were distinctly different from those of the thermophilic C. fetus ss. jejuni strains as shown both by COA and L-RIE. Serotypes A and B of the two former species were also differentiated. In COA tests the C. fetus ss. jejui=ni organisms gave the strongest reactions with homologous antibodies, but several interstrain cross-reactions were seen. By absorption strain specific COA reagents were obtained. Several reactions of identity indicating cross-reactive antigens were also seen with L-RIE within the ss. jejuni group. These results generally agreed with those obtained by COA. With the use of unheated, boiled or autoclaved organisms or sonicates heat labile antigens were differentiated from heat stable ones with the use of COA and R-LIE. The apparent antigenic heterogeneity of Campylobacteria indicates the importance of their serological grouping, e.g. for clinical and epidemiological investigations. COA and immunoelectrophoresis techniques can be effectively applied for such studies.

Serology of Neisseria gonorrhoeae. Characterization of rabbit hyperimmune antisera by line-rocket immunoelectrophoresis for use in coagglutination.

The co-agglutination (COA) and line-rocket immunoelectrophoresis (L-RIE) techniques were investigated for the serological classification of Neisseria gonorrhoeae. COA with absorbed sera was found to demonstrate "strain-specific" as well as cross-reacting antigens. The results were dependent on the immunizing, absorbing and agglutinating properties of a particular strain. Heated (100 degrees C) whole cells were found to be more reactive in the COA tests than untreated or formalin-fixed organisms. It was possible by L-RIE to compare the antigenic relationship between at least 20 strains in a single test run. This technique was applied on the 16 major outer membrane protein (MOMP) reference strains, which could then be divided into two separate groups with regard to cross-reacting antigens. Based on the findings by the L-RIE tests, selective absorptions of antibodies were performed for use in preparation of COA reagents. It was found that the reactions of these reagents with the MOMP reference strains could be assigned to three different antigenic classes. Reagents prepared for these classes are proposed as a basis for serological classification of Neisseria gonorrhoeae.

Serology of Neisseria gonorrhoeae. Demonstration by co-agglutination and immunoelectrophoresis of antigenic differences associated with colour/opacity colonial variants.

Serological classification of Neisseria gonorrhoeae by co-agglutination (COA) into previously described antigen classes W and J was confirmed in the present work. Immunization of rabbits with classified organisms gave antibodies which produced the expected results when used in the preparation of COA reagents. Colour/opacity colonial variants of isogenic strains, characterized by stereomicroscopy as opaque and transparent, respectively, were found to influence immunization, absorption, co-agglutination and precipitation in immunoelectrophoresis (IE). It was shown by COA and IE that organisms of opaque colonies often contained an extra antigenic factor(s) which resisted heating at 100 degrees C but was sensitive to treatment with pronase. They were extracted by heating the organisms in saline or lithium chloride solution. Serological classification with COA in clinical isolates was reproducible with reagents for antigen classes W and J. Colony morphology dependent reactions, mostly due to organisms of opaque colonies, occurred in 7% with reagents for antigen class W and in 20% for antigen class J.

SEROLOGY OF NEISSERIA GONORRHOEAE. CLASSIFICATION BY CO‐AGGLUTINATION

The co-agglutination (COA) method has been adapted for serological classification of Neisseria gonorrhoeae. COA reagents were prepared with selectively absorbed rabbit hyperimmune antibodies against gonoccal (GC) major outer membrane protein (MOMP) serotype strains. Using these reagents, the 16 MOMP reference strains could be referred to at least three antigen classes, tentatively named W, J and M. The GC antigens of class W were divided into three groups I, II and III, and they were in part sensitive to pronase. The antigens of class J reflected strain specific or serotype reactions, some sensitive and others resistant to proteolytic enzymes. The antigens of class M were sensitive to periodate and resistant to pronase. Strains used in serological studies by other authors were tested. The properties of class W correlated well with those of the so-called micro-immunofluorescence and immunotype systems, and class M with those of the so-called endotoxin and acid polysaccharide systems. Strains from three different laboratories could all be grouped by class W and M reagents. Identical strains obtained independently from different laboratories gave very similar reaction patterns with the reagents available. Repeated GC-isolates from patients infected with beta-lactamase producing strains showed stable reactions with class W and J reagents, while there was a time-related variation of the class M pattern. We have found that the COA method is rapid, easy and reproducible in the serological classification of Neisseria gonorrhoeae and all the 117 GC-strains tested could be classified.

Identification of Neisseria gonorrhoeae by the co-agglutination test (author's transl)

A slide co-agglutination test was perfomrd, using a serological identification reagent “Phadebact ® Gonococcus Test”(Pharmacia Diagnostic AB: Sweden) with 120 strains of Neisseria gonorrhoeae, 42 strains of N. meningitidis, 19 strains of other Neisseria species, 6 strains of Branhamella catarrhalis and 2 strains of Haemophilus influenzae.The Menck's direct colonies method, by which a few trypsin treated colonies of organisms grown on serum-free medium were mixed directly with the reagent, produced 10% of non-interpretable results among 80 strains of N. gonorrhoeae. All of the non-interpretable were predominated with T2 colony. In addition 11% of N. gonorrhoeae produced negative results by this method, and all the neagative strains were constituted of T4 colony only.On the other hand, a new procedure of boiling colonies method, described by the manufacturer, by which the organisms suspended in distilled water were heated at 100°C for 5 min prior to testing, produced positive results in all strains of N. gonorrhoeae examined. No positive results were obtained with 69 sti ains of non-gonococcal isolate by either method. However the number of non-interpretable was reduced and that of negative was increased by boiling colonies method.Using 120 strains of N. gonorrhoeae a comparison was made between co-agglutination (COA) test and conventional biological identification test by the Cystine Trypticase Agar (CTA) sugar degradation procedure. All strains of N. gonorrhoeae produced positive results by the COA test, while 2 strains 1.7% did not degrade glucose by the CTA procedure.The fresh clinical isolates as well as stock strains of N. gonorrhoeae produced positive results by the COA test, excepting for some few strains constituting of T4 colony only which produced somewhat weak postitive reaction.Using the colonies of oxidase positive and Gram-negative cocci primarily isolated on selective medium, or colonies transferred on non-selective media, a rapid identification by COA test alon was possible, except for a few non-gonococcal colonies with non-interpretable results which needed biological identification.Though there are some problem with respect to price and supply route for the reagent, the COA test is considered to be a very convenient, practical and accurate serological identification method for N. gonorrhoeae even for small bacteriological laboratories.

A COMPARISON OF FOUR METHODS FOR THE SEROTYPING OF GROUP B STREPTOCOCCI

Group B streptococci are implicated in a wide range of clinical conditions in human adults and neonates. The Group is subdivided into five serotypes Ia, Ib, Ic, II and III, which are differentiated on the basis of capsular polysaccharides. In the interests of epidemiology and efficiency a cheap, rapid method which is easily interpreted would be advantageous. In this study four methods of serotyping, namely, counter-immunoelectrophoresis (CIEP), microimmunodiffusion (MID), coagglutination (COA), and the Lancefield capillary precipitin (CP) test were compared in terms of ease of operation and interpretation, accuracy and rapidity. Todd Hewitt Broth (THB) cultures and acid extracts of the group B streptococcal strains were used as antigens for these methods. It was concluded that COA using THB cultures allows cheap and rapid screening for presumptive serotyping, having a 93-96% correlation with the CP test. MID gives an accurate (100% correlation with the CP test) and unambiguous confirmatory diagnosis of serotype.