Production of herpes simplex virus fluorescein labelled typing reagents.

29 herpes simplex virus monoclonal antibodies (MAbs) were produced and shown by indirect immunofluorescence assay (IIFA) to be HSV type specific or HSV cross reactive. Two HSV-1 and two HSV-2 specific MAbs were selected as HSV typing reagents on the basis of their specificity and the fluorescence pattern produced. When directly conjugated to fluorescein both reagents detected and correctly identified HSV isolates previously typed by restriction endonuclease analysis (REA). No cross reactions were observed with a panel of human viral pathogens. Assessment of these reagents in parallel with the Syva Microtrak Culture Identification/Typing Test indicated 100% concordance for the detection and typing of 80 clinical isolates.

An immune dot-blot technique for the diagnosis of ocular adenovirus infection.

The development of a monoclonal antibody based radio-immune dot-blot technique (IDBT) for the rapid detection of adenovirus is described. 718 conjunctival swabs from patients with acute keratoconjunctivitis were examined by conventional cell culture isolation techniques and IDBT. IDBT identified adenovirus in 64 of 75 culture positive samples and also in a further 34 culture negative samples [Sensitivity (IDBT versus culture) 85.3%; Specificity 92.2%]. IDBT is considered to be a simple, clinically relevant, technique for the rapid identification of adenovirus infection of the eye.

The impact of CE marking: is there a down side?

In summary, the IVDD was never intended to include in house assays produced within healthcare institutions, and it was clear to the authors that it would be impractical for them to comply with the Directive. The above points apply to all pathology specialties, all of which will be profoundly affected if the MHRA's interpretation of the Directive is upheld. It must be appreciated that, as well as affecting a large number of specialist and reference laboratories, many NHS laboratories perform in house assays on samples provided by other trusts. The costs involved in complying with the IVDD, if the present MHRA interpretation is upheld, will divert funds away from other healthcare initiatives and will inevitably stifle further development of specialist in house assays which make an immense contribution to our health services. The principles of the Directive are sound and its introduction will have a beneficial affect on pathology due to better regulation of IVDs, which are placed on the market. However, applying the Directive to in house assays can only be detrimental to the efficacy of pathology services across the UK and therefore have a negative affect on the health of the nation.

Monoclonal antibodies for the identification of herpesvirus simiae (B virus).

To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus). Mabs could be divided into groups A-E displaying specific reactivity for B virus (A); reactivity with both B virus and SA 8 but not HSV (B); reactivity with B virus, SA 8 virus and HSV strains (C); specific reactivity with HSV-1 (D); and specific reactivity with HSV-2 (E). Two of the B virus specific mabs were able to differentiate between cynomolgus and rhesus strains of B virus. None of the mabs reacted with human varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. A panel of mabs for the unequivocal identification of B virus isolates from monkey or man is proposed.

Development and preliminary evaluation of an enzyme immunosorbent assay for the detection of adenovirus type 8.

Epidemic keratoconjunctivitis (EKC) is a highly contagious adenoviral ocular infection which can occur in outbreaks and is predominantly caused by adenovirus type 8 (Ad8). Detection and typing of this virus after isolation is generally by serum neutralisation or more complex molecular techniques. These methods can be time consuming particularly during outbreaks. Therefore, an Ad8 specific antigen capture enzyme immunosorbent assay (EIA) was developed as a rapid and cost effective diagnostic method for laboratories. An Ad8 type-specific monoclonal antibody was used in a direct antigen capture method and an anti-hapten fluorescein isothiocyanate (FITC)-anti-FITC system. Assay configuration studies indicate that the system in which a type specific capture antibody and a group specific detector antibody are used provides greater sensitivity to the assay than a system using a group specific monoclonal or polyclonal capture antibody. Also, this allows the use of the same detector antibody with varying type specific capture antibodies on the solid phase. In a preliminary evaluation of the two formats, the direct antigen capture assay and the FITC-anti-FITC system had a sensitivity of 98.75% and 100%, respectively, with a specificity of 100%. However, these results are not statistically significant due to the low numbers of Ad8 and other viral isolates obtained from eye swabs. The direct EIA format has been shown to be able to detect different Ad8 genome types including four isolated from four epidemics of EKC in Brest, France, the Ad8 prototype strain, and some DNA-variant isolates which had not been typed by restriction endonuclease analysis (REA).

Preparation of monoclonal antibodies to JC virus and their use in the diagnosis of progressive multifocal leucoencephalopathy.

Seven monoclonal antibodies (MAbs) to polyomavirus JC were produced, and one was selected for use in immunofluorescence (IF) tests on brain material from patients with suspected progressive multifocal leucoencephalopathy (PML). The selected MAb (5.12.2) reacted by IF with JC-infected primary human foetal glial (PHFG) cell cultures (titre 1/200,000) but not with BK-infected human embryo lung (HEL) fibroblasts (titre less than 1/20). Its haemagglutination-inhibition (HI) titre was high (greater than 1/5 x 10(6)) against JC virus but low (less than 1/5) against BK virus. A diagnosis of PML was confirmed on brain biopsy or at postmortem in four patients, two of whom were also infected with human immunodeficiency virus (HIV). In one of the patients, specific detection of JC virus antigen had not been possible using our routine high titred JC and BK human sera due to interference by JC antibody present in the cerebrospinal fluid (CSF). Viral antigen was, however, detected with the MAb 5.12.2.

Rapid diagnosis of cutaneous herpes simplex infections using specific monoclonal antibodies.

One hundred children with suspected herpes simplex virus (HSV) infection and 20 controls were studied to compare a rapid immunofluorescence (RIF) test for detection and typing of HSV from smears of lesions with standard viral culture. The RIF test was evaluated for ease of use and speed of diagnosis. RIF and/or culture were positive in 64% of patients. All infections diagnosed by RIF and culture were HSV type 1. In 92% of patients RIF and culture results were in concordance. In 57 cases, RIF and cultures were positive for HSV infection and in 35 cases RIF and cultures were negative for HSV infection. Three patients had inadequate samples for RIF and five children had positive RIF but were culture negative. All controls had negative results both by RIF test and culture. The RIF test demonstrated 100% sensitivity and 95% specificity. The RIF test was type specific, easy to perform and gave diagnosis of HSV infections within an hour of taking the clinical specimen. This study suggests the RIF test is as good, if not more sensitive, in the diagnosis of HSV infections as standard viral culture and has the advantage of speed of diagnosis.

Rapid detection and serotyping of adenovirus by direct immunofluorescence.

Four fluorescent antibody reagents were evaluated for their suitability for the identification of adenovirus isolates by immunofluorescence. The antibodies used in the reagents consist of monoclonal antibodies against adenovirus type 3 (Ad3), Ad4, Ad8, and adenoviruses of subgroup C (Ad1,2,5,6), serotypes known to occur in outbreaks of disease. Most of the monoclonal antibodies employed were reactive against type-specific antigens found on the hexon protein. Reagents employing two noncompeting anti-hexon antibodies were more sensitive than reagents prepared with only one monoclonal antibody, although both types of reagents exhibited a high degree of specificity. Five hundred and seventeen adenovirus isolates (359 of which had previously been typed by other methods) and 46 nonadenovirus isolates were examined with all four type-specific reagents in parallel with an adenovirus group-specific reagent. The results indicate that direct typing of adenovirus isolates is feasible, leading to significant savings in time compared to other typing methods and should contribute to the management of certain adenovirus infections, particularly during outbreaks.