Use of urine albumin measurement as a replacement for total protein.

We have investigated the replacement of urine total protein estimations for the assessment of glomerular permeability, by the measurement of urine albumin excretion using a latex particle enhanced immunoturbidimetric assay. An initial screen was performed using Albustix to assess the sample pre-dilution necessary for immunoanalysis. A total of 167 24-hour urine samples were analysed and urine albumin concentration correlated well with that of urine total protein (r = 0.93) over the range 0-16,800 mg/l. This protocol provides a more cost effective and analytically valid assessment of glomerular permeability.

Targeting and degradation of p53 by E6 of human papillomavirus type 16 is preferential for the 1620+ p53 conformation.

E6-mediated degradation of p53 is believed to play a role in the transformation of cells by high-risk types of human papillomavirus. In order to explore the structural requirements for targeting of p53 we have compared E6-mediated degradation of variant p53 forms expressed in vitro. Complete degradation was observed in samples containing monomers, dimers and higher molecular weight structures of wild-type p53, indicating that E6 targets all quaternary forms of wild-type p53. Wild-type human and murine p53s reactive with PAb 1620 (which recognizes a conformation-dependent epitope) were degraded when incubated with E6. Mutant p53 proteins were variably resistant to E6-mediated degradation, and this correlated with PAb 1620 reactivity. Thus, mutants hp53Val-154, hp53Val-266 and hp53Pro-273 (1620 degrees) were completely resistant to degradation, whereas hp53Ile-247 and hp53Trp-248 (1620+) were degraded. Mutants hp53Leu-273 and mp53Val-135, which are temperature sensitive for conformation, were completely degraded in the 1620+ form but degradation resistant in the 1620 degrees form. Although the PAb 1620+ conformation appeared important for recognition of p53 by E6, the epitope itself is unlikely to be the actual recognition target since the PAb 1620 monoclonal antibody failed to protect against E6-mediated degradation.

Partially transformed T3T3 cells express high levels of mutant p53 in the 'wild-type' immunoreactive form with defective oligomerization.

High levels of wild-type p53 suppress transformed growth of many cell lines and yet murine T3T3 cells shown partially transformed growth despite high endogenous levels of phenotypically 'wild-type' p53. On sequencing T3T3 p53 was found to encode missense mutations at codons 230 and 287 and, although endogenous T3T3 p53 is 'wild type', the protein adopted the mutant phenotype when expressed in vitro. Size fractionation of T3T3 cell lysate indicated monomeric p53 possibly in complex with a low molecular weight protein. When expressed in vitro T3T3 p53 formed dimers and higher order structures. Thus T3T3 cells appear (i) to drive endogenous mutant p53 to adopt conformational epitopes characteristic of the 'wild-type' protein, and (ii) to interfere with normal assembly of p53 quaternary structure. Phosphopeptide mapping of p53 from 3T3x cells, T3T3 cells and SV3T3 cells indicated reduced amino terminal phosphorylation of the mutant p53 phenotype. Alternative splicing of p53 was also detected in 3T3x cells; similar splicing occurs in wild-type p53 (Han & Kulesz-Martin, 1992; Nucl. Acids Res., 20, 1979-1981) and a possible regulatory function is discussed.

Temperature-sensitive mutants of p53 associated with human carcinoma of the lung.

We have compared the effects of specific point mutations on the tertiary and quaternary structure of the human p53 protein. Eight mutants, each derived from primary resected tissues of lung carcinomas, were expressed in vitro under strictly defined conditions, such that the only known variant was the point mutation present in each p53 mRNA. All the mutations were located in highly conserved domains. The tertiary structure of each mutant protein was investigated by reactivity with anti-p53 monoclonal antibodies directed against conformation-dependent epitopes. Quaternary structure was examined by gel filtration. Although all the mutant proteins exhibited abnormal tertiary structures, their quaternary structures appeared similar to wild type, the one exception being p53-tyr135, which contains tyrosine in place of cysteine at residue 135. The conformational phenotype of mutant human p53 was found to be dependent upon (i) the locus of the mutation and (ii) the nature of the amino acid substitution: two different substitutions at residue 273 yielded two mutants with differing structural properties. We have discovered three mutants of human p53 that are temperature sensitive for conformation; one is mutated at codon 273, a 'hotspot' for p53 mutation in human cancer.

Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles.

Turbidimetric immunoassay is commonly used to quantify serum proteins. Latex-particle enhancement of this type of assay has been primarily associated with increasing assay sensitivity. However, covalent coupling of an antibody to a latex particle can offer other advantages that are also pertinent in measurement of high concentrations of analytes. By using a common antibody with IgG as a model analyte, we describe the development of a nonenhanced and a latex-particle-enhanced turbidimetric assay for measuring serum IgG. Both assays show adequate analytical recovery and parallelism, and results compare well with those by rate nephelometry. The latex-enhanced assay has equivalent sensitivity, working range, and interassay precision, but much greater signal change and calibration stability than the nonenhanced assay. In addition, with latex particles, less antiserum is needed. Coupling antibodies to latex particles offers considerable advantages, even when an improved assay detection limit is not required.

Development of a rapid latex enhanced turbidimetric assay for retinol binding protein in urine.

We describe a simple, rapid and sensitive homogeneous immunoassay for urinary retinol-binding protein (RBP) using latex particle-enhanced turbidimetric immunoassay. Rabbit anti-human RBP is covalently coupled to 40 nm latex particles and the assay performed on the IL Monarch 2000 centrifugal analyser, with a 20 microL sample volume and the reaction monitored at 340 nm over an 8 min period. The assay range is 0-6 mg/L with a detection limit of 25 micrograms/L. The within and between assay coefficients of variation are less than 1.5% and less than 2.5%, respectively. Comparison with radioimmunoassay for RBP showed good agreement.

Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation.

Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.

Particle-enhanced turbidimetric immunoassay of sex-hormone-binding globulin in serum.

A particle-enhanced turbidimetric immunoassay (PETIA) for human sex-hormone-binding globulin (SHBG) is described. The method involves use of antibody covalently coupled to latex particles and is almost fully automated, with sample processing being complete in less than 20 min. The working reagents are stable for at least three months, and full calibration of the assay each day is not essential. A particular advantage is that pretreatment of samples is rarely required because the working range of the assay is from 2.0 to 320 nmol/L for nondiluted serum. Intra- and interassay CVs were less than 4.5% and 8.5%, respectively, and mean analytical recovery was 101.5%. SHBG concentrations of 129 serum samples determined by this method and by a commercially available immunoradiometric assay correlated highly.

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135.

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.

A rapid and robust particle-enhanced turbidimetric immunoassay for serum beta 2 microglobulin.

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination.

Rapid, robust method for measuring low concentrations of albumin in urine.

We describe a rapid particle-enhanced turbidimetric immunoassay for albumin in urine. Intra- and interassay CVs were less than 5% and less than 10%, respectively, the detection limit is 2 mg/L, and the working range extends to 200 mg/L. Mean analytical recovery of albumin added to centrifuged urines was 100% (SD 10.6%), and, when results were compared with those by the Pharmacia RIA, the correlation coefficient was 0.99. The working reagents are stable for at least six months; thus this assay is suited for both batch and urgent analysis.