ENZYME IMMUNOASSAY FOR DETECTION OF RUBELLA SPECIFIC IgM ANTIBODY

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  • 394

    RESULTS

    Values are meansSEM. *p

  • 395

    Fig. I-Rubella specific IgM response detected by an enzyme IgMcapture assay in sera from individuals after recent and pastinfection and from controls.

    Controls include a group of rheumatoid factor (RF) positive individuals,shown separately.

    1=rubella specific IgM confirmed.Absorbance values=OD492 test minus OD492 standard seronegative serum.

    for detecting IgM antibodies to hepatitis A).5 We have investigatedan enzyme ACCA for the detection of rubella specific IGM.Polystyrene Removastrip wells (Dynatech) were coated with an

    immunoglobulin preparation of sheep anti-human-IgM (50 g/ml) in 0-05molll "tris" buffer pH 7-5 5 and then incubated with test samples of humanserum diluted 1/200 in 2% bovine serum albumin/saline for 3 h at 37C. Afterthe wells had been washed, excess rubella haemagglutinin antigen was addedand allowed to stand at 37C for 4 h. After a further wash, the working dilutionof horseradish peroxidase labelled anti-rubella conjugate prepared byperiodate oxidation and diluted in 20% sheep serum/saline was added to thewells and incubated for 2 h at 37C. The amount of conjugate bound wasdetermined by adding o-phenylenediamine/urea peroxide and measuring theabsorbance at 492 nm.

    The results obtained with 28 samples taken from twenty-fiveadult females at various intervals following rubella infection, 15samples from individuals with high rubella IgG alone, 15 samplesfrom individuals with rubella IgG and rheumatoid factor, and 11seronegative individuals are shown in fig. 1. Absorbance valuesabove those obtained with the control groups were observed in thesamples taken after acute infection and as early as one day after thereported onset of a rash. Rubella specific IgM was confirmed by anindependent test (closed circle) in all these sera: The meanabsorbance value for the control rheumatoid factor positive groupwas marginally greater than that observed with the seronegativesamples. A limited number of post-vaccination samples have alsobeen tested; a proportion were positive in our assay but the levels ofIgM present were considerably less than those found followingnatural infection. Our failure to detect specific IgM in all post-vaccination sera may be due to the limiting test sensitivity or mayreflect a failure to obtain samples during the period of peakresponse.The sensitivity of our capture assay has been compared with that

    obtained in indirect immunofluorescence tests performed on frac-tionated sera and the results (fig. 2) indicate comparable sensitivity

    7. Duermeyer W, Wielaard F, van der Veen J. A new principle for the detection of specificIgM antibodies applied to an ELISA for hepatitis A. J Med Virol 1979; 4: 25-32.

    I,"T I,IV I,U"T If"" " .,,"--.-

    Immunofluorescence Titre

    Fig. 2-Comparison of rubella specific IgM measured by enzymecapture assay and indirect immunofluorescence on fractionatedsera.

    with a correlation coefficient of 0-88. Further investigations are inprogress to examine the limit of sensitivity of our assay.We thank Dr J. Best and Dr J. Cradock-Watson for the supply of test samples

    and access to clinical data.

    Wellcome Research Laboratories,Beckenham, Kent BR3 3BS

    JOHN A. DIMENTSHIREEN M. CHANTLER

    PLASMA-TRIFLUOPERAZINE CONCENTRATIONSDURING HIGH DOSE THERAPY

    SIR,-Trifluoperazine has been used in psychiatry since 1958, butthere is no published information on its plasma concentrations. 1,2,Existing neuroleptic drug assays are unsuitable for thiscompound.3,4 A high pressure liquid chromatographic (HPLC)method for trifluoperazine has been developed by one ofus (S.H.C.)and we now report detection and measurement of trifluoperazine inthe plasma of a single patient receiving high doses.The patient, an 18-year-old man, entered hospital during a schizo-

    phrenic episode within a recurrent illness. In an attempt to deter-mine suitable treatment, he was first treated with trifluoperazineliquid concentrate at a dose of 15 mg x 3 daily. This was later changedto 30 mg and then to 80 mg, both at bedtime. No trifluoperazine wasdetected in his plasma at the two lower doses, but the concentrationsgiven in the table were recorded after an 80 mg test dose, given onemorning as a dose additional to the normal daily 80 mg regimen.Blood was collected into heparinised tubes and the trifluoperazine was ex-

    tracted into hexane from alkalinised plasma. The concentrated extractswere examined by HPLC using a Micropak-CN column (Varian), a mobilephase of methanol (90%)/5 mmolll ammonium acetate (10%) at 2 - 5 5 ml/mm,and ultraviolet (254 nm) detection. Standards (spiked plasma) and patientsamples were handled similarly. The calibration equation was of the formy=a+bx (r2) where y is instrument response in absorbance units, a is the in-tercept on the y axis, b is the slope, x is the plasma concentration of triflu-operazine in ng/ml, and r is the square of the correlation coefficient:

    y=(-l-071xlo")+(2-46xlo

    1. van Praag HM. Psychotropic drugs: a guide for the practitioner. London: Macmillan,1978.

    2. Cooper TB. Plasma level monitoring of antipsychotic drugs. Clin Pharmacokin 1978;3: 14-38.

    3. Curry SH, Marshall JHL. Plasma levels of chlorpromazine and some of its relativelynon-polar metabolites in psychiatric patients Life Sci 1968, 7: 9

    4 Curry SH Determination of nanogram quantities of chlorpromazine and some of itsmetabolites in plasma using gas-liquid chromotography with an electron-capturedetector Analyt Chem 1968, 40: 1251-55

    5. Butterfield AG, Sears RW. High-performance liquid chromatographic determinationof perphenazine and amitriptyline hydrochloride in two-component tablet formula-tions. J Pharm Sci 1977; 66: 1117-19.

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