Dhanraj Samuel

Development of a quantitative immuno-PCR assay and its use to detect mumps-specific IgG in serum.

Determination of the immune status of individuals to vaccine-preventable diseases requires an assay that can detect antibodies that may be present at very low levels, especially when natural or vaccine exposure may have been many years previously. Immuno-PCR (iPCR) has recently been described as an ultrasensitive method for the detection of antigens and we have adapted the method for the quantification of antibodies to mumps virus. The procedure used was similar to an indirect ELISA except that the detecting antibody (anti-human IgG) was chemically conjugated to a short capture oligonucleotide rather than an enzyme. The capture oligonucleotide was then detected by the addition of target DNA, which was designed to hybridise to the capture oligonucleotide and function as a template for real-time PCR. The quantity of target DNA detected by the PCR depended upon the level of specific antibody in the test sample. We found that the sensitivity (and specificity) of the iPCR assay did not exceed that of the conventional ELISA. The sensitivity was limited by nonspecific binding of human IgG to the solid phase. Further development of reagents and assay formats is necessary to fully exploit the potential of quantitative iPCR, so that potential improvements in the sensitivity of anti-mumps IgG detection can be realised.

Development of a measles specific IgM ELISA for use with serum and oral fluid samples using recombinant measles nucleoprotein produced in Saccharomyces cerevisiae

In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein. Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter. High levels of rMVN (20 mg/litre of yeast culture) were generated. Electron microscopy showed that the purified rMVN assembled into typical herring-bone structures. Monoclonal antibodies produced to the rMVN also reacted with native measles virus N in immunofluorescence tests. The purified rMVN and a monoclonal antibody to the rMVN conjugated to horseradish peroxidase were used to develop a measles specific IgM capture EIA (MACEIA) in both serum and oral fluid specimens. Evaluations of the MACEIA were performed by testing a) serum samples (n=80) and b) paired oral fluid/serum samples from measles cases (n=50, representing 16 cases) and oral fluids from controls with non-measles rash (n=59, representing 48 cases). The samples were also tested for measles IgM, using a reference radioimmunoassay (MACRIA). The sensitivity and specificity of the MACEIA compared with MACRIA for a) the serum samples were 100 and 96.6% respectively and b) for paired serum/oral fluids samples 100 and 100%, respectively.

A quantitative immuno-PCR assay for the detection of mumps-specific IgG

Sensitive assays are required for seroprevalence studies of measles, mumps and rubella (MMR)-vaccinated populations where many may have low levels of antibodies. This protocol describes a quantitative immuno-PCR assay to detect mumps-specific IgG antibodies. The purpose of the protocol is to determine the immune status of individuals to mumps. Mumps-specific IgG from a dilution of patients serum is bound by recombinant mumps nucleoprotein coated on the surface of microtitre plate wells. Bound antibody is detected by PCR using a conjugate of anti-human IgG covalently coupled to an oligonucleotide. The oligonucleotide is detected by the addition of target DNA, designed to hybridise to the oligonucleotide and serve as a template for real-time PCR using the LightCycler. The quantity of target DNA detected by the PCR depends upon the level of specific antibody in the test sample.

A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC

Monoclonal antibodies to FITC were produced and shown to be specific for the fluorochrome. Molecular weight marker proteins labelled with FITC could be detected after SDS-PAGE and transfer onto nitrocellulose using anti-FITC followed by an anti-mouse IgG-alkaline phosphatase conjugate. The molecular weight of an antigen common to Legionella pneumophila and recognised by a monoclonal antibody could be determined accurately on a Western blot when FITC labelled markers were used as internal standards. The FITC-anti-FITC system was shown to be extremely sensitive, detecting 23.7 amol of BSA-FITC conjugate (equivalent to 1.42 x 10(7) molecules of FITC) in a dot blot assay.

A comparison of the binding of biotin and biotinylated macromolecular ligands to an anti-biotin monoclonal antibody and to streptavidin.

A competitive enzyme immunoassay was used to study the binding of biotinylated macromolecular ligands and d-biotin to an anti-biotin monoclonal antibody and to streptavidin. Solid phase BSA-c-biotin competed with biotin or biotinylated macromolecular ligands in solution for receptor binding. The concentration of d-biotin required to inhibit streptavidin binding to solid phase BSA-c-biotin by 50% was 11.5 pM. This streptavidin-biotin interaction was taken as having an affinity/avidity index of 100 and all other receptor-ligand interactions were calculated relative to this. The avidity indices calculated for streptavidin interactions with BSA-c-biotin and IgG-biotin were 17.6 and 6.6 respectively, whereas for anti-biotin the values for these ligands were 20.5 and 19.9 respectively. The interaction of anti-biotin with d-biotin had an affinity index of 0.001. Although streptavidin has the greatest binding affinity for d-biotin, its avidity for biotinylated ligands was considerably lower and comparable to that observed for anti-biotin-biotinylated macromolecule interactions.

The FITC-anti-FITC system is a sensitive alternative to biotin-streptavidin in ELISA.

In a model ELISA system alkaline phosphatase (AP) absorbed onto microtitre wells was employed as the target antigen. The antigen was then reacted with a monoclonal antibody to AP either unlabelled or labelled with (a) FITC and (b) biotin. The bound anti-AP was then detected with horseradish peroxidase (HRP) conjugates of polyvalent anti-mouse IgG, and the FITC and biotin anti-AP conjugates with HRP conjugates of a monoclonal anti-FITC or streptavidin respectively. The FITC-anti-FITC system proved to be of similar sensitivity to the biotin-streptavidin system detecting 140 amol compared to 350 amol of antigen. Both these methods of antigen detection were superior to the anti-mouse IgG reagent (2100 amol). In contrast to biotinylated antibodies, FITC-labelled antibodies are highly coloured and fluorescent. These features aid the preparation, purification and characterisation of conjugates. In addition, very low non-specific binding is encountered with enzyme conjugates of anti-FITC and this may confer an advantage over enzyme conjugates of avidin/streptavidin reagents.

An amplified ELISA for the detection of parvovirus B19 IgM using monoclonal antibody to FITC

A new M-antibody capture ELISA for the detection of specific IgM against parvovirus B19 is described. The test uses a monoclonal anti-fluorescein isothiocyanate (FITC) antibody conjugated to horseradish peroxidase to amplify the positive reactions. Serum samples (N = 823) submitted for B19 IgM assay were tested in parallel in the new ELISA and the standard B19 M-antibody capture radioimmunoassay (MACRIA) test. By both tests B19 IgM was detected in 38 (4.6%) samples, and not detected in 771 (94%) samples. One sample was positive in the ELISA test but negative in the RIA and of the 13 sera giving equivocal results in the MACRIA, 6 were positive and 7 negative. If the RIA equivocal results were excluded, the ELISA showed 100% (38/38) sensitivity and 99.9% (771/772) specificity compared to the MACRIA test. The B19 IgM ELISA is a sensitive and specific test with better discrimination between anti B19 IgM positive and negative specimens than MACRIA.

Synthesis of mumps virus nucleocapsid protein in yeast Pichia pastoris.

The expression of mumps virus nucleocapsid protein in yeast Pichia pastoris was investigated. Viral nucleocapsid proteins usually elicit a strong long-term humoral immune response in patients and experimental animals. Therefore, the detection of antibodies specific to mumps virus nucleoprotein can play an important role in immunoassays for mumps diagnosis. For producing a high-level of recombinant mumps virus nucleoprotein the expression system of yeast P. pastoris was employed. The recombinant nucleocapsid protein was purified by cesium chloride ultracentrifugation of yeast lysates. Electron microscopy of the purified recombinant nucleocapsid protein revealed a herring-bone structure similar to the one discovered in mammalian cells infected with mumps virus. The yield of purified nucleocapsid-like particles from P. pastoris constituted 2.1 mg per 1 g of wet biomass and was considerably higher in comparison to the other expression systems.

A microtitre plate method for isolation and typing of poliovirus using a Blue-Cell ELISA

A simple, sensitive, specific and rapid procedure for isolating and typing polioviruses is described. Specimens are inoculated onto confluent monolayers of cell lines (Hep-2C, L20B or RD) seeded into microtitre plates. After 24-48 h, the infected cells are stained with monoclonal antibodies specific for poliovirus types 1,2,3 or a blend of the three antibodies followed by an anti-mouse IgG-horseradish peroxidase conjugate. On addition of substrate, infected cells stain an intense blue colour and are easily distinguished from uninfected cells by light microscopy. Poliovirus infection can be detected before the appearance of cytopathic effects (CPE). This Blue-Cell ELISA test was evaluated against conventional culture and seroneutralisation on a range of polio isolates and clinical specimens. The sensitivity and specificity of the Blue-Cell ELISA compared to neutralisation was 100% (87/87) on culture supernatants of poliovirus isolates sent to our reference laboratory for confirmation. All the poliovirus isolates were typed within 24 h of specimen inoculation using the new method compared to 6-10 days by conventional culture and neutralisation. The method proved to be more sensitive than conventional culture when clinical specimens were examined. Of 43 clinical specimens from which poliovirus had been previously isolated by various laboratories in the U.K., 30/43 (69.8%) were positive for poliovirus by the Blue-Cell ELISA compared to 29/43 (67.4%) by conventional culture and neutralisation. Neutralisation of specimens exhibiting CPE indicated that all of the polioviruses were correctly typed with the new method. CPE was not observed by conventional culture in any specimen that was negative in the Blue-Cell ELISA. There were no cross-reactions with a range of other enteroviruses.

A single-step method for the purification of anti-FITC antibodies by use of a coumarin immunosorbent

Monoclonal anti-fluorescein isothiocyanate (FITC) antibody cross-reacts with 7-hydroxy coumarin derivatives conjugated to BSA. This property permitted the affinity purification of monoclonal anti-FITC antibodies from ascitic fluid using an-immunosorbent consisting of a 7-hydroxy coumarin derivative linked to Sepharose 4B. Ascitic fluid was applied to the immunosorbent column and, after washing, the bound antibody was eluted under extremely mild conditions using 3 M MgCl2. Antibody eluted in this manner was greater than 96% pure as assessed by SDS-PAGE. A polyclonal sheep anti-FITC antibody was also purified from serum on the same immunosorbent to greater than 94% purity. This simple and rapid method for the purification of anti-FITC antibodies will find applications in both immunodiagnostic procedures and in studies of hapten-antibody interactions. The affinity constant of the purified monoclonal anti-FITC antibody conjugated to horseradish peroxidase was assessed by ELISA and was found to be 1.5 x 19(9) M-1.