The effect of dodecyl sulfate on immunoglobulin hapten binding.

The instantaneous effect of dodecyl sulfate (DDS), in the mM concn range, on the binding of monovalent hapten by immunoglobulin was examined. Fluorescence measurements were utilized to study the effect of the detergent on sheep antiserum generated against thyroxin (T4) and against methamphetamine. Haptens were conjugated with the thiocyanate derivative of fluorescein in order to determine hapten binding on the basis of increased fluorescence polarization for the fluorescein-thiocarbamyl-hapten adducts (FT4 or FA) bound to immunoglobulin. Incubation of anti-T4-serum with DDS for 1 hr before the addition of FT4 resulted in diminished binding. The effect occurred at DDS concns greater than 0.1 mM and was essentially complete at a DDS conc of 1 mM. A kinetic study demonstrated a two stage process. An initial, rapid stage, with a half time less than 30 sec accounted for a reduction of immunoglobulin binding by 75%. The remaining 25% binding capacity was lost during a second, much slower phase with a half-time of about 11/2 hr. Prior hapten binding inhibited the effect of DDS. The degree of protection from combining site denaturation afforded by prior hapten binding was limited by the dissociation rate of bound hapten. The major, rapid phase was completely and immediately reversible by dilution. Prolonged incubation in DDS resulted in irreversible denaturation. The overall rate of DDS denaturation of the entire immunoglobulin molecule, as revealed by changes in the circular dichroism spectrum of a sheep gamma globulin fraction, was considerably slower than the denaturation rate of the combining site.

Characteristics of prostatic infarcts and their effect on serum prostate-specific antigen and prostatic acid phosphatase.

OBJECTIVES: To determine how prostatic infarcts affect serum prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) levels. METHODS: Two hundred eighteen clinically benign, whole prostates were obtained at autopsy, completely sectioned, and examined histologically. PSA and PAP levels were determined from premortem serum. RESULTS: Six of the 218 (2.8%) prostates had infarcts. The infarcts were usually multiple and usually located in the central and/or middle concentric zones of the middle third of the prostate without a preference for a particular lobe. Serum PSA by immunoradiometric assay were elevated in all 6 cases. Serum PAP by both enzymatic assay (ACA), and immunoradiometric assay were available for 5 cases and were elevated by both methods in 2 cases, approached elevated levels by both methods in 1 case, and were normal by both methods in 2 cases. The PSA and PAP levels appeared to be affected more by the age than by the size of the infarct. CONCLUSIONS: Prostatic infarcts elevate PSA levels more frequently than PAP levels, and prostatic infarcts may be responsible for some unexplained elevations of serum PSA and PAP levels.

Homogeneous, micelle quenching fluoroimmunoassay for detecting amphetamines in urine.

We developed a homogeneous fluoroimmunoassay for detecting amphetamines in urine. Only fluorescence intensity need be measured because the emission of non-protein-bound fluorescein-labeled amphetamine is preferentially quenched by detergent micelles. In a previous reported prototype assay system for measuring gentamicin in serum we used fluorescein and dodecyl sulfate (Anal Chem 1985; 57:1928-30). We have found that favorable hydrophobic and (or) ionic character of the analyte and unfavorable polar and (or) ionic character of the fluor are important determinants of the desired interactions. An anionic detergent and fluorescein, therefore, should be appropriate for apolar of cationic analytes, such as gentamicin and amphetamines. A greater [H+] at the anionic micelle surface is important for quenching emission from the fluor moiety. Millimolar concentrations of dodecyl sulfate rapidly denature immunoglobulin unless hapten is bound with sufficiently high affinity. Affinity was sufficiently high for the antibody used in the prototype gentamicin assay but not for the amphetamine antibody. Thus for the amphetamine assay, we used a non-denaturing detergent, dodecyl(oxyethylene)12 sulfate. The assay requires 30 microL of specimen in 2 mL of total assay volume. Amphetamine(d-,dl-, and meth-), at a concentration of 1 mg per liter of urine, is readily detected.

Characterization and mathematical correction of hemolysis interference in selected Hitachi 717 assays.

The effect of hemolysis on several assays performed with the Hitachi 717 was quantified by relating the amount of error to the concentration of hemoglobin. Hemolysis interference was judged clinically significant when analyte concentration varied by > 10% from the initial value. Hemolysis interference was significant for alkaline phosphatase, aspartate aminotransferase, alpha-amylase, bilirubin, creatine kinase, gamma-glutamyltransferase, lactate dehydrogenase, lactate dehydrogenase-1, potassium, and theophylline assays. Error (expressed in absolute terms) was linearly dependent on hemoglobin concentration and independent of the initial analyte concentration in each case, except for bilirubin and theophylline, where multiple regression analysis was required to quantify the effect. Relative error was dependent on the initial analyte concentration in all cases. Correction formulas were calculated from linear regression of absolute error vs hemoglobin concentration. Clinical application of correction formulas and mechanisms of hemolysis interference for each assay are discussed.

Prostatic acid phosphatase levels (enzymatic method) from completely sectioned, clinically benign, whole prostates.

Clinically benign, whole untrimmed prostates were obtained from 104 patients at autopsy, completely sectioned, and examined microscopically. The histological and gross findings of the prostate were correlated with premortem prostatic acid phosphatase levels (PAP, enzymatic method, ACA, Dupont Co.) to determine how often carcinoma of the prostate (CAP) affected PAP levels and to identify other findings within the prostate associated with elevated PAP levels. Sixty (58%) prostates did not have CAP, 34 (33%) had CAP smaller than 1 ml in volume, and 10 (10%) had CAP larger than 1 ml in volume. PAP levels were elevated (greater than 1 U/L) in 8 of 60 (13%) prostates without CAP, in 2 of the 34 (6%) prostates with CAP smaller than 1 ml, and in 1 of the 10 (10%) prostates with CAP larger than 1 ml. These differences were not statistically significant. Likewise, a statistically significant correlation between PAP levels and patient age, patient race, severe inflammation, of high grade prostatic intraepithelial neoplasia (PIN) was not found. However, there was a statistically significant correlation between PAP levels and prostate weight (p < 0.0001). This study suggest that PAP cannot distinguish between patients with clinically undetected CAP and patients without CAP. Furthermore, elevated PAP levels are often not due to metastatic CAP and additional evidence should be present, even in patients with known CAP, before an elevated PAP level is considered to be conclusive evidence of metastatic CAP.