CLICK-FLISA Based on Metal-Organic Frameworks for Simultaneous Detection of Fumonisin B1 (FB1) and Zearalenone (ZEN) in Maize.

Mycotoxins are secondary products produced primarily by fungi and are pathogens of animals and cereals, not only affecting agriculture and the food industry but also causing great economic losses. The development of rapid and sensitive methods for the detection of mycotoxins in food is of great significance for livelihood issues. This study employed an amino-functionalized zirconium luminescent metal-organic framework (LOF) (i.e., UiO-66-NH2). Click chemistry was utilized to assemble UiO-66-NH2 in a controlled manner, generating LOF assemblies to serve as probes for fluorescence-linked immunoassays. The proposed fluoroimmunoassay method for Zearalenone (ZEN) and Fumonisin B1 (FB1) detection based on the UiO-66-NH2 assembled probe (CLICK-FLISA) afforded a linear response range of 1-20 µmol/L for ZEN, 20 µmol/L for FB1, and a very low detection limit (0.048-0.065 µmol/L for ZEN; 0.048-0.065 µmol/L for FB1). These satisfying results demonstrate promising applications for on-site quick testing in practical sample analysis. Moreover, the amino functionalization may also serve as a modification strategy to design luminescent sensors for other food contaminants.

Establishment of a microspheres-based homogeneous fluorescence immunoassay for the rapid detection of cardiac troponin I.

Myocardial infarction occurs rapidly, and thus the rapid detection of cTnI levels is the key to its diagnosis. Most current assays take 10-30 min. In this study, we developed a method for accurately measuring cardiac troponin I (cTnI) levels in human sera with amplified luminescence neighborhood homogeneous assay (AlphaLISA). The method involves coupling two cTnI antibodies targeting different epitopes to the surface of carboxylated donor and acceptor beads. The final signal values were detected by the double-antibody sandwich method, and the best reaction conditions were obtained by optimizing the experimental conditions. The sensitivity, specificity, accuracy, and precision of the method were evaluated. Results showed that the method requires only 3 min to produce the results, the detection sensitivity is 27.06 ng L-1, and the measurement range is 34.56-62 500 ng L-1. cTnI-AlphaLISA has an intra-assay precision of 2.18-4.57% (<10%) and an inter-assay precision of 5.60-6.95% (<10%). The relative recovery rates are within reasonable limits. In addition, the serum assay results of the method were compared with chemiluminescence immunoassay, and the results are in agreement with one another (ρ = 0.8803; P < 0.0001). The method is expected to be developed as a routine method, but further studies and evaluations are needed.

Development of an immunoassay for quantification of soluble human CD40L (CD154) in plasma and serum samples.

When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu3+-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis.

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

A sensitive fluoroimmunoassay for quantitative detection of imidacloprid based on quantum dot-streptavidin conjugate.

Imidacloprid (IMI), the most commonly used neonicotinoid, is widely present in both the environment and agro-products due to extensive and prolonged application, posing potential risks to ecological security and human health. This study introduced a sensitive and rapid fluorescence-linked immunosorbent assay, employing Quantum Dot-Streptavidin conjugate (QDs-SA-FLISA), for efficient monitoring of IMI residues in agro-products. Under optimized conditions, the QDs-SA-FLISA exhibited a half-maximal inhibition concentration (IC50) of 1.70 ng/mL and a limit of detection (LOD, IC20) of 0.5 ng/mL. Investigation into the sensitivity enhancement effect of the QDs-SA revealed that the sensitivity (IC50) of the QDs-SA-FLISA was 7.3 times higher than that of ELISA. The recoveries and relative standard deviation (RSD) ranged from 81.7 to 118.1 % and 0.5-9.4 %, respectively, for IMI in brown rice, tomato and pear. There was no significant difference in IMI residues obtained between QDs-SA-FLISA and UHPLC-MS/MS. Thus, the QDs-SA-FLISA represents a reliable approach for the quantitative determination of IMI in agro-products.

Clinical impact of thyroglobulin (Tg) and Tg autoantibody (TgAb) measurements in needle washouts of neck lymph node biopsies in the management of patients with papillary thyroid carcinoma

ABSTRACT Objectives The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. Materials and methods We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patient’s serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. Results The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. Conclusions Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant.

Reibergrama para evaluar la síntesis intratecal de Lectina de unión a Manosa / Reibergram to evaluate the Mannan binding Lectin intrathecal synthesis

INTRODUCCIÓN: la dinámica particular de las proteínas derivadas del cerebro en el líquido cefalorraquídeo es diferente a la dinámica de las proteínas derivadas de la sangre. OBJETIVO: describir los datos empíricos de la lectina de unión a manosa y brindar una interpretación teórica de la dinámica de esta proteína a través de la confección un nuevo reibergrama. MÉTODOS: la lectina de unión a manosa en suero y líquido cfalorraquídeo, fue medida en 40 adultos normales a través de un ensayo inmunofluorométrico. El criterio diagnóstico estuvo basado en; muestras controles (pacientes normales) y muestras de pacientes con enfermedades que cursaron con disfunción de barrera sangre-líquido cefalorraquídeo. RESULTADOS: el coeficiente de correlación entre la lectina de unión a manosa en el líquido cefalorraquídeo y en el suero, fue muy bajo. El reibergrama de la lectina de unión a manosa se diseñó de acuerdo con procedimientos previos. CONCLUSIONES: bajo cualquier condición de barrera sangre-líquido cefalorraquídeo, el reibergrama puede identificar la ocurrencia de síntesis intratecal de lectina de unión a manosa.

BACKGROUND: The dynamics of brain derived proteins in cerebrospinal fluid is different from the dynamics of blood-derived proteins. Aim: To describe the empirical data for mannan binding lectin and gives a theoretical interpretation of the dynamics of this protein in cerebrospinal fluid through a new reibergram. METHODS: Serum and cerebrospinal fluid mannan binding lectin were measured in 40 normal adults by immunofluorometric assays. The diagnostic criteria were based in; normal control samples defined clinically and diseases with blood-cerebrospinal fluid barrier dysfunction. RESULTS: Correlation coefficient between cerebrospinal fluid MBL and serum MBL was very low. Mannan binding lectin reibergram was designed according with previous procedures. CONCLUSION: Under all conditions of the blood-cerebrospinal fluid barrier, the reibergram can identify the occurrence of intrathecal mannan binding lectin synthesis.

Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin / Desenvolvimento, caracterização e validação clínica de um novo ensaio sensível para a dosagem da tiroglobulina sérica

OBJECTIVE: In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS: The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS: The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION: This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

OBJETIVO: Na última década, estudos mostraram a importância dos ensaios de tiroglobulina (Tg) com melhor sensibilidade funcional no seguimento dos pacientes com carcinoma diferenciado de tiroide (CDT). Neste estudo, descrevemos o desenvolvimento de um novo ensaio de Tg de alta sensibilidade, que foi validado no seguimento de pacientes com CDT e correlacionado com um ensaio comercialmente disponível. SUJEITOS E MÉTODOS: O ensaio imunofluorométrico de Tg baseia-se em anticorpos, um monoclonal e um policlonal desenvolvidos em nosso laboratório. Avaliamos 100 amostras de soro de 87 pacientes com CDT submetidos à tiroidectomia total, sendo que 87% deles também receberam 131I. A Tg foi dosada também em ensaio comercial (Beckman Access). RESULTADOS: A correlação entre os dois métodos foi de 0,74 (p < 0,0001). O novo ensaio mostrou uma sensibilidade funcional de 0,3 ng/mL. A sensibilidade diagnóstica foi de 88,9%, que aumentou para 100% quando associada ao ultrassom cervical (US). CONCLUSÃO: O novo método de dosagem de Tg mostra boa correlação com o ensaio comercial Beckman Access e com a evolução clínica dos pacientes. O novo ensaio será fundamental no seguimento dos nossos pacientes com CDT. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

Weight-adjusted neonatal 17OH-progesterone cutoff levels improve the efficiency of newborn screening for congenital adrenal hyperplasia / Determinação de pontos de corte para 17OH-progesterona de acordo com o peso ao nascimento melhora a eficiência da triagem neonatal da hiperplasia adrenal congênita

OBJECTIVE: To evaluate weight-adjusted strategy for levels of neonatal-17OHP in order to improve newborn screening (NBS) efficiency. SUBJECTS AND METHODS: Blood samples collected between 2-7 days of age from 67,640 newborns were evaluated. When N17OHP levels were > 20 ng/mL, and a second sample was requested. We retrospectively analyzed neonatal-17OHP levels measured by Auto DELFIA- B024-112 assay, grouped according to birth-weight: G1: < 1,500 g, G2: 1,501-2,000 g, G3: 2,000-2,500 g and G4: > 2,500 g. 17OHP cutoff values were determined for each group using the 97.5th, 99th, 99.5th and 99.8th percentiles. RESULTS: 0.5 percent of newborns presented false-positive results using the cutoff level > 20 ng/mL for all groups. Neonates of low birthweight made up 69 percent of this group. Seven full-term newborns presented congenital adrenal hyperplasia (CAH) and, except for one of them, 17OHP levels were > 120 ng/mL. Only the 99.8th percentile presented higher predictive positive value (2 percent), and lower rate of false-positives in all groups. CONCLUSIONS: We suggest the use of 99.8th percentile obtained by weight-adjusted N17OHP values of healthy newborns to reduce the rate of false-positive results in NBS.

OBJETIVO: Avaliamos retrospectivamente os valores da 17OHP ajustados para o peso ao nascimento para melhorar a eficiência da triagem neonatal. SUJEITOS E MÉTODOS: 67.640 recém-nascidos com amostras coletadas entre 2-7 dias de vida. Uma segunda amostra foi solicitada na presença de testes com valores da 17OHP > 20 ng/mL. 17OHP dosada pelo método DELFIA- B024-112 e correlacionada com o peso ao nascimento: G1 < 1.500 g, G2 1.501-2.000 g, G3 2.000-2.500 g e G4 > 2.500 g. Pontos de corte da 17OHP foram determinados para cada grupo usando os percentis 97,5th, 99th, 99,5th e 99,8th. RESULTADOS: Falso-positivos ocorreram em 5 por cento dos resultados com o ponto de corte > 20 ng/mL, dos quais 69 por cento eram prematuros. Sete recém-nascidos apresentaram deficiência da 21-hidroxilase e, exceto em um, os valores da 17OHP foram > 120 ng/mL. Somente o valor da 17OHP do 99,8th apresentou maior valor preditivo positivo (2 por cento) e menor índice de falso-positivos. CONCLUSÕES: Sugerimos o valor da 17OHP do 99,8th ajustado para o peso ao nascimento para se reduzir a taxa de resultados falso-positivos da triagem neonatal.

Heterogeneity of carboxyl-terminal parathyroid hormone circulating forms in patients with hyperparathyroidism due to end stage renal disease / Heterogeneidade das formas carboxi-terminal circulantes de paratormônio em pacientes com hiperparatiroidismo devido à insuficiência renal crônica terminal

OBJECTIVE: To study carboxyl-terminal (COOH) parathyroid hormone (PTH) circulating forms in patients with hyperparathyroidism due to end stage renal disease (ESRD). METHODS: An immunometric assay that recognizes both intact and COOH PTH forms was developed. The assay, in conjunction with an intact assay, was used to measure PTH in serum samples obtained from 25 patients with hyperparathyroidism due to ESRD. Samples were also submitted to gel filtration chromatography in a Superdex® 30 1.6 x 60 cm column, and the PTH content in the elution tubes, measured using both assays. RESULTS: Values from 39.000 to 232.300 ng/mL (mean ± sd = 101.680 ± 45.330 ng/mL) were found using the COOH assay (PTH 39-84 was used as standard). Values obtained by the intact PTH assay ranged from 318 to 3.307 ng/mL (1.769 ± 693 ng/mL) with a correlation between assays of 0.462 (p = 0.02). The elution profile obtained using the COOH assay showed a preponderance of forms with MW ranging from 8.500 to 4.500 daltons. The profiles obtained from the 25 patients were very similar. CONCLUSIONS: In patients with hyperparathyroidism due to ESRD circulating PTH levels contain a broad range of molecular forms including COOH with MW ranging from 8.500 to 4.500 daltons. These forms are not recognized by the standard intact PTH assays. The correlation of these findings to the clinical aspects of bone disease in ESRD patients remains to be studied.

OBJETIVO: Estudar as formas carboxi-terminal (COOH) circulantes de paratormônio (PTH) em pacientes com hiperparatiroidismo devido à insuficiência renal crônica (IRC) terminal. MÉTODOS: Foi desenvolvido um ensaio imunométrico que reconhece formas intactas e COOH longas de PTH. Esse ensaio foi utilizado, em conjunto com um ensaio para molécula intacta de PTH, em amostras de 25 pacientes com hiperparatiroidismo devido à IRC. As amostras também foram submetidas à cromatografia de gel filtração em coluna de Superdex® 30 de 1,6 x 60 cm, e o conteúdo de PTH nos tubos de eluato foi medido, empregando-se os dois ensaios. RESULTADOS: Valores entre 39.000 e 232.300 ng/mL (média ± dp = 101,680 ± 45,330 ng/mL) foram obtidos usando-se o ensaio COOH (PTH 39-84 foi utilizado como padrão). Com o ensaio para PTH intacto, os valores distribuíram-se entre 318 e 3,307 ng/mL (1,769 ± 693 ng/mL) com correlação entre ambos de 0,462 (p = 0,02). O perfil cromatográfico obtido com o ensaio COOH mostrou predomínio de formas com PM entre 8.500 e 4.500 daltons. Os perfis cromatográficos dos 25 pacientes foram bastante semelhantes. CONCLUSÕES: Em pacientes com hiperparatiroidismo devido à IRC, os níveis circulantes de PTH contêm um espectro de formas moleculares que incluem formas carboxi-terminais, com PM entre 8.500 e 4.500 daltons. Essas formas não são reconhecidas pelos ensaios de rotina utilizados para a medida de PTH intacto. A correlação entre esses achados e os aspectos clínicos da doença óssea em pacientes com IRC necessita de maiores estudos.

Determinación de sensibilización alérgica a dermatofagoides en niños de 5 años y menores por fluoroinmuno ensayo-UniCAP / Dust mite allergic sensitization in children aged 1 month to 5 years-old using specific IgE determination by unicap fluoroimmunoassay

Dust mite sensitization is considered a major risk factor for pediatric asthma; however, there are few data about early sensitization in Chilean children available. This study aimed to investigate allergic sensitization to mites in infancy and early childhood. The patient population, 224 children aged 0 to 5 years with suspected respiratory allergy, was divided into 3 groups of age: 1 to 12 months-old, 13 to 35 months-old, and 3 to 5 years-old. Sensitization status was ascertained by assessing the specific IgE to mite by UniCAP fluoroimmunoassay (Pharmacia). Sixty eight (30.6 percent) patients were sensitized to mites. The frequency and level of sensitization increased with the age of the children. Our data support that avoidance measures in the domestic environment aimed at the primary prevention of mite-driven sensitization should be introduced at the earliest possible stage.

La sensibilización a dermatofagoides es un factor de riesgo para el desarrollo de asma. Hay escasa información sobre la sensibilización temprana en niños chilenos. Objetivo: Investigar la sensibilización a dermatofagoides en niños de 5 años y menores. Método: Revisión de resultados de IgE específica por ensayo UniCAP (Pharmacia) contra dermatofagoides de niños de hasta 5 años con sospecha de alergia respiratoria, divididos en 3 grupos de edad: 1-12 meses, 13-35 meses y 3-5 años. Resultados: Se estudió un total de 224 niños, entre 1 mes y 5 años de edad (x 2,65 años +/- 1,48 DS), 57,1 por ciento de sexo masculino. Su distribución según grupo etario fue: 66 en el grupo 1; 45 en el grupo 2 y 113 en el grupo 3. El 30,6 por ciento presentó sensibilización a dermatofagoides (18,2 por ciento grupo 1; 33,3 por ciento grupo 2 y 36,3 por ciento grupo 3). El grupo 3 presentó concentraciones de IgE específica significativamente más elevadas que los otros grupos. Conclusiones: La sensibilización a dermatofagoides es detectable en menores de un año, con frecuencia y nivel que aumentan en forma paralela a la edad.

Concentração sérica de paratormônio intacto em cães com insuficiência renal crônica / Serum concentration of intact parathormone in dogs with chronic renal failure

Estudou-se a ocorrência de hiperparatireoidismo secundário renal e determinaram-se as concentrações séricas de paratormônio intacto (PTHi-c), cálcio total e fósforo em 30 cães com insuficiência renal crônica (IRC) e em 40 cães sadios. Para a determinação do PTHi-c, foi utilizado o método imunofluorométrico, com o emprego de anticorpos anti-aminoterminal (extraídos de gema de ovo da galinha) e de anticorpos monoclonais anti-carboxiterminal (H5P10), marcados com Europium. As concentrações séricas de PTHi-c (717,23±469,13pg/ml no grupo IRC e 36,76±34,40pg/ml no grupo-controle; P=0,0001), cálcio total (11,46±2,03mg/dl no grupo IRC e 10,11±0,91mg/dl no grupo-controle; P=0,003) e fósforo (12,01±8,06mg/dl no grupo IRC e 4,33±0,74mg/dl no grupo-controle; P=0,0001) foram mais altas nos cães com IRC. Observou-se estreita correlação entre PTHi-c e fósforo (r=0,56; P=0,0006), o que não ocorreu entre PTHi-c e cálcio. Hipercalcemia em cães com alta concentração de PTHi-c demonstrou a possível ocorrência de hiperparatireoidismo terciário em 11 animais. A hiperfosfatemia pode indicar, de forma indireta, a ocorrência de hiperparatireoidismo secundário nos cães com IRC.

The occurrence of renal secondary hyperparathyroidism was studied and serum intact parathormone (PTHi-c), total calcium and phosphorus were measured in thirty dogs with chronic renal failure (CRF) and in forty healthy dogs. The imunnofluorometric method was used for the PTHi-c assay, using anti-aminoterminal antibodies (obtained from chicken yolk) and anti-carboxiterminal monoclonal antibodies (H5P10), marked with Europium. Mean value ± SD of serum concentration of PTHi-c was 717.23±469.13pg/ml in CRF group and 36.76±34.40pg/ml in control group (P=0.0001); for total calcium it was 11.46±2.03mg/dl in CRF group and 10.11±0.91mg/dl in control group (P=0.003); and for phosphorus it was 12.01±8.06mg/dl in CRF group and 4.33±0.74mg/dl in control group (P=0.0001). The highest values were observed in CRF dogs. A positive correlation between PHTi-c and phosphorus was observed (r=0.56; p=0.0006), and no correlation was detected between PTHi-c and total calcium. In dogs with CRF, hypercalcemia in presence of high level of serum PTHi-c showed a possible indication of tertiary hyperparathyroidism in 11 animals. In conclusion, hyperphosphatemia could indirectly demonstrate the occurrence of secondary hyperparathyroidism in CRF dogs.