MATERIALES REQUERIDOS PERO NO PROVISTOS

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TSH ELISA R6 RC REFERENCIAS

1. Frank JE; Faix JE; Hermos RJ; Mullaney DM; Rojan DA; Mitchell ML; Klein RZ Thyroid function in very low birth weight infants: effects on neonatal hypothyroidism screening. J Pediatr 1996;128(4):548-54.

2. Thakur C; Saikia TC; Yadav RN. Total serum levels of triiodothyronine (T3) thyroxine (T4) and thyrotropine (TSH) in school going children of Dibrugarh district: an endemic goitre region of Assam. Indian J Physiol Pharmacol 1997;41(2):167-70.

3. Morimoto K; Inouye K.A sensitive enzyme immunoassay of human thyroid-stimulating hormone (TSH) using bispecific F(ab')2 fragments recognizing polymerized alkaline phosphatase and TSH. J Immunol Methods 1997;205(1):81-90.

4. Maes M; Mommen K; Hendrickx D; Peeters D; D'Hondt P; Ranjan R; De Meyer F; Scharp´e S. Components of biological variation, including seasonality, in blood concentrations of TSH, TT3, FT4, PRL, cortisol and testosterone in healthy volunteers. Clin Endocrinol (Oxf) 1997;46(5):587-98.

2014-07-07

Cat#: TS227T (96 Tests) Por pedidos y/o consultas técnicas por

favor contactar a: Calbiotech Inc., 10461 Austin Dr, Spring Valley, CA, 91978

Tel (619) 660-6162, Fax (619) 660-6970, www.calbiotech.com

Inmuno-ensayo para la Determinación Cuantitativa de la Concentración

de Tirotropina (TSH) en suero humano ELISA

Número de Catálogo TS227T (96 Tests) INTENCION DE USO:

El kit TSH ELISA de Calbiotech Inc. se utiliza para la determinación cuantitativa de Tirotropina (TSH) u Hormona Estimulante de la Tiroides, en suero o plasma humano.

RESUMEN

La Tirotropina (TSH) es una hormona glicoproteica secretada por el lóbulo anterior de la hipófisis (adenohipófisis) y regula la secreción de Triiodotironina (T3) y de Tiroxina (T4), por parte de la Tiroides. La TSH cuenta con dos subunidades llamadas Alfa y Beta. La subunidad Alfa es similar a las subunidades alfa que se encuentran en otras hormonas glicoproteicas tales com LH, FSH y hCG. Asimismo, la subunidad Beta resulta específica y difiere de entre hormona y hormona. La medición de TSH en suero es una de las herramientas más importantes para el diagnostico de desórdenes en la glándula tiroidea. Niveles elevados de TSH en sangre resultan en un sensible y temprano indicador de hipotiroidismo. Bajos niveles de TSH resultan indicadores de hipertiroidismo. La sensibilidad del kit TSH de Calbiotech Inc. es de 0.05 µlU/ml.

PRINCIPIO DEL ENSAYO

TSH es un método Elisa en fase sólida sandwich. Las muestras, el conjugado Anti-TSH-HRP/Biotina, se añaden a los pocillos designados recubiertos con Streptavidina. El TSH en el suero del paciente forma una reacción tipo sándwich entre dos anticuerpos específicos de TSH. La proteína y el conjugado HRP no unidos son lavados por la solución de lavado. Tras la adición del sustrato, la intensidad del color es proporcional a la concentración de TSH en las muestras. Una curva estándar se prepara sobre la intensidad del color a la concentración de la TSH.

MATERIALES PROVISTOS 96 Pruebas

1. Micropozos recubiertos con Streptavidina 12x8x1 2. Estándares de TSH: 7 viales (listos para su uso) 0.5ml 3. Conjugado Reactivo TSH: 1 frasco (listo para su uso) 12ml 4. Sustrato TMB: 1 frasco (listo para su uso) 12ml 5. Solución de Frenado: 1 botella (listo para su uso) 12ml 6. Solución de Lavado Concentrado 20X: 1 frasco 25ml

MATERIALES REQUERIDOS PERO NO PROVISTOS 1. Agua destilada o desionizada.

2. Pipetas de precisión. 3. Puntas de pipetas desechables.

4. Lector Microelisas con lente a 450nm de longitud de onda con una banda de amplitud de 10nm o menor y un rango de densidad óptica de 0-2 OD ó mayor

5. Papel absorbente o toalla de papel. 6. Papel cuadriculado.

ALMACENAMIENTO 1. Almacene el kit a 2 - 8°C.

2. Mantenga las tiras de los pocillos selladas en la bolsa de aluminio.

3. Todos los compuestos son estables hasta su fecha de expiración siempre y cuando las condiciones de almacenaje sean estrictamente llevadas a cabo como aquí se indica.

4. No exponga los reactivos al calor, luz solar o intensa luz eléctrica. CUIDADOS Y PRECAUCIONES

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TSH ELISA R6 RC 1. Potencial de los materiales de riesgo biológico: Los calibradores contienen componentes de origen humano, que

se han sido probados y encontrados no reactivos para el antígeno de superficie de hepatitis B y anticuerpos contra el VIH Aprobado por la FDA. Sin embargo no hay método de prueba que puede ofrecer completa seguridad de que el virus VIH, Hepatitis B u otros agentes infecciosos estén presentes. Estos reactivos deben ser manejados según el Nivel de Bioseguridad 2, como se recomienda en los Centros para el Control de Enfermedades / Institutos Nacionales de Salud manuales. "Bioseguridad en laboratorios microbiológicos y biomédicos” 1984.

2. No pipetee con la boca. No fume, coma, o beba en el área donde maneje este equipo.

3. Los componentes en este equipo son para uso como una unidad integral. Los componentes de diferentes lotes no se deben mezclar.

4. Es recomendable que los estándares, controles y muestras de suero se corran por duplicado.

5. Para obtener óptimos resultados, debe apegarse estrictamente al protocolo. Pipeteado exacto y preciso, así como después de la hora exacta y requerimientos de temperatura prescritos son esenciales. Cualquier desviación de este puede resultar en datos no válidos.

RECOLECCION DE LA MUESTRA

1. Recolecte sangre por venopunción y separe el suero de inmediato.

2. En caso de no llevar a cabo el examen inmediatamente, refrigere la muestra a (2-8º C) por cinco días. En caso de exceder dicho plazo, congele a -20º C hasta un mes.

3. Evite múltiples ciclos de congelamiento-descongelamiento de la muestra. 4. Previo al ensayo, la muestra deberá ser debidamente descongelada y mezclada. 5. Evite utilizar muestras con exceso de lípidos.

PREPARACION DEL REACTIVO

Preparar solución de lavado a 1x adicionando 475ml de agua destilada o desionizada al frasco de (25ml a 20x). Guarde a temperatura ambiente.

PROCEDIMIENTO DEL ENSAYO

Previo al ensayo, permita que todos los reactivos alcancen la temperatura ambiente (18°-26°C). Mezcle suavemente los reactivos antes de su uso.

1. Corte el número de pozos a utilizar para que realizar el ensayo en duplicado para cada muestra. Cierre y selle el resto de los micro pocillos no utilizados y refrigérelos a 2-8° C.

2. Vierta 50 µl de estándares de TSH, especímenes y controles en los pozos apropiados.

3. Vierta 100 µl del conjugado reactivo TSH en todos los pocillos. Agite gentilmente la microplaca por 20-30 segundos para mezclar los reactivos.

4. Cubra e incube a temperatura ambiente por 60 minutos (18-26°C).

5. Retire el líquido de los pocillos. Enjuague y lave los pocillos tres veces con 300 µl de solución de lavado de 1X. Golpee la placa de los micro pocillos sobre el papel absorbente para remover las gotas de agua residuales. 6. Vierta 100 µl de sustrato TMB en todos los pocillos.

7. Cubra e incube a temperatura ambiente por 15 minutos.

8. Frene la reacción agregando 50 µl de solución de frenado a cada pozo. Sacuda gentilmente para facilitar el mezclado de la solución por 15-20 segundos.

9. Lea la densidad óptica a 450nm con un lector de placa de micro valoración en un plazo de 15 minutos después de haber agregado la solución de frenado.

CALCULO DE RESULTADOS

La curva estándar se construye de la siguiente manera:

1. Compruebe el valor estándar de TSH en cada vial estándar. Este valor puede variar de lote a lote. Asegúrese de verificar el valor de cada kit.

2. Para construir la curva estándar, trazar la absorbancia para el estándar de TSH (eje vertical) frente a las concentraciones estándar de TSH (eje horizontal) en un papel gráfico lineal. Dibuje la mejor curva a través de los puntos.

3. Lea la absorbancia de los controles y cada muestra desconocida. Registre el valor de cada control o muestra desconocida.

EJEMPLO DE CURVA STANDARD

OD 450 nm Conc. pg/mL Std 1 0.033 0 Std 2 0.062 0.5 Std 3 0.21 2.5 Std 4 0.41 5 Std 5 0.75 10 Std 6 1.37 20 Std 7 2.61 40 VALORES EXPERADOS

Se recomienda que cada laboratorio establezca sus propios rangos normales sobre la base de una muestra representativa de la población local. Los siguientes valores de TSH pueden ser utilizados únicamente como guia:

Clasificacion Rango Normal µlU/ml

Adultos 0.4-4.2

Recien Nacidos 1.0-3.9

2 a 20 semanas 1.7-9.0

21 semanas a 20 años 0.7-6.4

LIMITACIONES DEL TEST

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ACTH ELISA R5 RC QUALITY CONTROL

Control plasma or plasma pools should be analyzed with each run of calibrators and patient samples. Results generated from the analysis of the control samples should be evaluated for acceptability using appropriate statistical methods. In assays in which one or more of the quality control sample values lie outside the acceptable limits, the results for the patient sample may not be valid.

LIMITATIONS OF THE PROCEDURE

The CBI ACTH ELISA kit has exhibited no “high dose hook effect” with samples spiked with 20,000 pg/ml of ACTH. Samples with ACTH levels greater than the highest calibrator, however, should be diluted and reassayed for correct values.

EXPECTED VALUES

ACTH levels were measured in eighty-three (83) apparently normal individuals. The values obtained ranged from 7.9 to 66.1 pg/ml. The geometric mean + 2 standard deviations of the mean were calculated to be 8.3 to 57.8 pg/ml. It’s recommended that each lab establishes its own normal range.

REFERENCES

1. Ryan, WG: Endocrine Disorders – A Pathophysiologic Approach, 2nd Edition Year Book Medical Publishers, Inc. 1980.

2. Watts, N.B., J.H. Keffer: Practical Endocrine Diagnosis, Third Edition, Lea and Febioer, 1982.

3. Ganong, WF. L.D. Alber, TC Lee: ACTH and the Regulation of Adrenocorticol Secretion, N. Engl. J. Med. 290 : 1006, 1974.

4. Tepperman, J: Metabolic and Endocrine Physiology, 4th Edition, Year Book Medical Publishers, Inc.,1981. 5. Odell, W.D., R. Horton, M.R. Pandian, J. Wong: The Use of ACTH and Cortisol Assays in the Diagnosis of

Endocrine Disorders. Nichols Institute Publication, 1989.

6. Radioimmunoassay Manual, Edited by A.L. Nichols and J.C. Nelson, 4th Edition Nichols Institute, 1977. 7. Gold, E.M.: The Cushing’s Syndromes: Changing Views of Diagnosis and Treatment. Ann Intern. Med. 90:829,

1979.

8. Plasma Cortisol, RIA for Physicians, Edited by J.C. Travis, 1:8, Scientific Newsletter, Inc. 1976. 9. Krieger, D.T.: Physiopathology of Cusihing’s Disease, Endocrine Review 4:22-43, 1983.

2014-07-07

Cat#: AC018T(96 Tests) For Order and Inquiries, please contact

Calbiotech Inc.,

10461 Austin Dr, Spring Valley, CA, 91978 Tel (619) 660-6162, Fax (619) 660-6970,

www.calbiotech.com

Adrenocorticotropic Hormone (ACTH) ELISA

Catalog No. AC018T (96 Tests)

INTENDED USE

The Calbiotech ACTH ELISA Kit is intended for the quantitative determination of ACTH Adrenocorticotropic Hormone) in human plasma. For research use only.

SUMMARY AND EXPLANATION

ACTH is a 39-amino acid peptide hormone (MW=4500) secreted by the pituitary to regulate the production of steroid hormones by the adrenal cortex. ACTH increases the synthesis and release of all adrenal sterioids, aldosterone, cortisol and adrenal androgens. It is the principal modulator of cortisol, the most important glucocorticoid in man. As the cortisol level in blood increases, release of ACTH is inhibited directly at the pituitary level. Through this same mechanism, decreasing cortisol levels lead to elevated ACTH levels. In healthy individuals, ACTH reaches a peak in the early morning (6:00 - 8:00 hour) and levels become lowest late in the day and near the beginning of the sleep period. Stress may also override the diurnal variation. Plasma ACTH assays are useful in the differential diagnosis of pituitary Cushing’s disease, Addison’s disease, autonomous ACTH producing pituitary tumors (e.g. Nelson’s syndrome), hypopituitarism with ACTH deficiency and ectopic ACTH syndrome. Primary adrenocortical insufficiencies, Addison’s disease. Hypopituitarism with ACTH deficiency, which is secondary adrenocortical insufficiency, is characterized by low plasma ACTH and cortisol concentrations, and a subnormal, but usually distinct adrenal response to stimulation with synthetic ACTH (Cortrosyn).

PRINCIPLE OF THE TEST

The CBI ACTH Immunoassay is a two-site ELISA for the measurement of the biologically active 39 amino acid chain of ACTH. One antibody is prepared to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared to bind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with HRP for detection. In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the TMB substrate. Stop solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of ACTH in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of ACTH present in the controls and patient samples are determined directly from this curve.

MATERIALS PROVIDED 96 Tests Microwells coated with Streptavidin 12x8x1 Biotinylated ACTH Antibody (Reagent 1) 2.7 ml Peroxidase (Enzyme) labeled ACTH Antibody (1 Vial) 2.7 ml Wash Concentrate (1 Vial) 30 ml TMB Substrate (1 Vial) 15 ml

Stop Solution (1 Vial) 20 ml

Calibrators (5 Vials) 2 ml

Zero Calibrator (1 Vial) 4 ml Controls 1 & 2 (CTRL) (2 Vials) 2 ml

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ACTH ELISA R5 RC MATERIALS NOT PROVIDED

1. Distilled or deionized water 2. Precision pipettes 3. Disposable pipette tips

4. ELISA reader capable of reading absorbance at 450 nm 5. Absorbance paper or paper towel

6. Graph paper

STORAGE AND STABILITY 1. Store the kit at 2 – 8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose test reagents to heat, sun or strong light. WARNINGS AND PRECAUTIONS FOR USERS

1. Potential biohazardous materials:

The calibrator and controls contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984.

2. This kit is designed for research use only.

3. Optimal results will be obtained by strict adherence to the test protocol. Precise pipetting as well as following the exact time and temperature requirements is essential.

4. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.

5. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.

6. This product contains components preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water. SPECIMEN COLLECTION & HANDLING

1. The determination of ACTH should be performed on EDTA plasma. 2. To assay the specimen in duplicate, 400 μl of EDTA plasma is required. 3. Collect whole blood in a lavender [EDTA] tube.

4. The plasma should be promptly separated, preferably in a refrigerated centrifuge, and stored at -20°C or lower.

5. EDTA plasma samples may be stored up to 8 hours at 2-8°C. 6. EDTA plasma samples frozen at -20°C are stable for up to 4 months. REAGENT PREPARATION AND STORAGE

Store all kit components at 2-8°C except Wash Concentrate and Stop Solution

1. All reagents except the non-zero calibrators, kit controls and the Wash Concentrate are ready-to-use. Store all reagents at 2-8°C, except the Wash Concentrate, which should be kept at room temperature until dilution to avoid precipitation.

2. For each of the non-zero calibrators (Calibrator B through F) and kit controls 1 and 2, reconstitute each vial with 2 ml of distilled or deionized water and mix. Allow the vial to stand for 10 minutes and then mix thoroughly by gentle inversion to insure complete reconstitution. Use the calibrators and controls as soon as possible upon reconstitution. Freeze (-20°C) the remaining calibrators and controls as soon as possible after use. Calibrators and controls are stable at -20°C for 6 weeks after reconstitution with up to 3 freeze thaw cycles when handled as recommended in “Procedural Notes” section

3. ELISA Reagent A: Wash Concentrate: Mix contents of wash concentrate thoroughly. If precipitate is present in the Wash Concentrate due to storage at lower temperature such as 4°C, dissolve by placing the vial in a 37°C water bath or oven with swirling or stirring. Add wash concentrate (30 ml) to 570 ml of distilled or deionized water and mix. The diluted working wash solution is stable for 90 days when stored at room temperature.

ASSAY PROCEDURE

1. Bring all specimens and kit reagents to room temperature (18-26 °C) and gently mix.

2. Place sufficient Streptavidin Coated Strips in a holder to run all six (6) ACTH calibrators, A - F of the ACTH CALIBRATORS (concentration is stated on the vial label), Quality Control Plasma and patient samples.

3. Pipet 200 μl of sample into the designated or mapped well. Freeze (-20°C) the remaining calibrators and controls as soon as possible after use.

4. Add or dispense 25 μl of Reagent 1 (Biotinylated Antibody) into each of the wells which already contain the sample.

5. Add or dispense 25 μl of Reagent 2 (Enzyme Labeled Antibody) into each of the same wells. Cover the microplate(s) with aluminum foil or a tray to avoid exposure to light, and place it on an orbital shaker or rotator set at 170 + 10 rpm for 4 hours + 30 minutes at room temperature (18-26°C). 6. First aspirate the fluid completely and then wash/aspirate each well five (5) times with the Working

Wash Solution (prepared from Reagent A), using an automatic microplate washer. The wash solution volume should be set to dispense 0.35 ml into each well.

7. Add or dispense 150 μL of the ELISA Reagent B (TMB Substrate) into each of the wells.

8. With appropriate cover to avoid light exposure, place the microplate(s) on an orbital shaker or rotator set at 170 + 10 rpm for 30 +5 minutes at room temperature (18-26°C).

9. Add or dispense 100 μl of the Stopping Solution into each of the wells. Mix gently.

10. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm against 250 μl of distilled or deionized water. Read the plate again with the reader set to 405 nm against distilled or deionized water. Note: The second reading is designed to extend the analytical validity of the calibration curve to the value represented by the highest calibrator, which is approximately 500 pg/ml. Hence, patient samples with ACTH > 150 pg/ml can be quantified against a calibration curve consisting of the readings all the way up to the concentration equivalent to the highest calibrator using the 405 nm reading, away from the wavelength of maximum absorbance. In general, patient and control samples should be read using the 450 nm for ACTH concentrations up to 150 pg/ml. ACTH concentrations above 150 pg/ml should be interpolated using the 405 nm reading. 11. By using the final absorbance values obtained in the previous step, construct a calibration curve via

cubic spline, 4 parameter logistics, or point-to-point interpolation to quantify the concentration of the ACTH.

CALCULATION OF RESULTS

1. For the 450 nm readings, construct a dose response curve (calibration curve) using the first five calibrators provided, i.e. Calibrators A, B, C, D and E. For the 405 nm readings, construct a second dose response curve using the three calibrators with the highest concentrations, i.e. Calibrators D, E and F.

2. Assign the concentration for each calibrator stated on the vial in pg/ml. Plot the data from the calibration curve on linear graph paper with the concentration on the X-axis and the corresponding A.U. on the Yaxis.

3. Draw a straight line between 2 adjacent points. This mathematical algorithm is commonly known as the "point-to-point" calculation. Obtain the concentration of the sample by locating the absorbance unit on the Y-axis and finding the corresponding concentration value on the X-axis. Patient and control samples should be read using the 450 nm for ACTH concentrations up to 150 pg/ml. ACTH concentrations above 150 pg/ml should be interpolated using the 405 nm reading.

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Androstenedione R3 RC The cross-reaction of the antibody calculated at 50% method, according to Abraham, are shown in the table:

Analyte % Cross reactivity

Androstenedione 100 Testosterone 0.3486 5 alpha-Dihydrotestosterone <0.0001 Androsterone 0.009 DHEA-S 0.0007 Cortisol <0.0001 17α Estradiol <0.0001 Estrone 0.0167 Androsterone-SO4 0.0017 Progesterone 0.1091 Desoxycorticosterone 0.200 REFERENCES

1. Horton R., Tait J., Androstenedione production and interconversion rates measured in peripheral blood and studies on the possible site of its conversion to testosterone.. J.Endocrinol Invest. 45: 301-313, 1966.

2. Dorfman RI., Shipley RA., Androgens. John Wiley and Sons, New York, 116-128, 1956

3. Erickson GF 1993 Normal regulation of ovarian androgen production. Seminars in Reproductive Endocrinology 11:307-312.

4. Kicman, A. T., Bassindale, T., Cowan, D. A., Dale, S., Hutt, A. J., and Leeds, A. R., Effect of androstenedione ingestion on plasma testosterone in young women; a dietary supplement with potential health risks Clin.Chemistry 2003; 49:167-169.

5. Brown, G.A., Vukovich, M.D., Martini, E.R., Kohut, M.L., Franke, W.D., Jackson, D.A., and King, D.S. Endocrine responses to chronic androstenedione intake in 30- to 56-year-old men. J Clin Endocrinol Metab 2000, 85:4074-4080.

2014-07-07

Cat#: AD183E (96 Tests) For Order and Inquiries, please contact

Calbiotech Inc., 10461 Austin Dr, Spring Valley, CA, 91978

ANDROSTENEDIONE ELISA

Catalog No. AD183E (96 Tests)

INTENDED USE

The Calbiotech, Inc. (CBI) Androstenedione ELISA Kit is intended for the measurement of Androstenedione in serum or plasma.For research use only.

Androstenedione is the primary precursor of testosterone in women. It is synthesized in the adrenal gland. Measurement of Androstenedione may be used as an indicator of androgenic activity in women. The steroid hormone Androstenedione is one of the main androgens, besides Testosterone and Dehydroepiandrosterone. In males, androgens are secreted primarily by the Leydig cells of the testes, to some degree also in the adrenal cortex. In females, the androgens are secreted mainly in the adrenal glands and in the ovary. Around 10% of the androgens are derived from peripheral conversion, mainly of DHEA. Androstenedione and Testosterone show high diurnal variability. The highest levels are measured in the morning. At the age of puberty serum androstenedione levels rise, after menopause they decline again. High androstenedione levels are measured during pregnancy. In women, high levels of androstenedione (47-100% above normal) are generally found in hirsutism, mostly in combination with other androgens as testosterone and DHEA-S. Androstenedione overproduction is due to ovarian dysfunction or maybe of adrenal origin. High circulating androstenedione levels are found in women with polycystic ovaries and 21-hydroxylase effect. Significant lower androstenedione levels are found in postmenopausal osteoporosis.

The CBI Androstenedione ELISA kit is based on the principle of competitive binding between Androstenedione in the test specimen and Androstenedione-HRP conjugate for a constant amount of rabbit anti-Androstenedione. In the first incubation, goat anti-rabbit IgG-coated wells are incubated with 25μl of Androstenedione standards, patient samples, 50μl Androstenedione-HRP conjugate reagent and 50μl rabbit anti-Androstenedione reagent at room temperature for 60 minutes. During the incubation, HRP labeled Androstenedione competes with the endogenous Androstenedione in the standard and sample, for a fixed number of binding sites of the specific Androstenedione antibody. Thus, the amount of Androstenedione peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of Androstenedione in the specimen increases. Unbound Androstenedione peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 15 minutes, resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450nm. A standard curve is prepared relating color intensity to the concentration of Androstenedione.

MATERIALS PROVIDED 96 Tests

1. Microwells coated with Goat anti-rabbit IgG 12x8x1 2. Standard : 6 vials (ready to use) 0.5 ml 3. Enzyme Conjugate (ready to use) 7 ml 4. Rabbit Anti- Androstenedione Reagent (ready to use) 7 ml 5. TMB substrate (ready to use) 12 ml 6. Stop solution (ready to use) 12 ml 7. Wash Solution 20x Concentrated 25 ml

MATERIAL NOT PROVIDED

4. ELISA reader capable of reading absorbance at 450nm 5. Absorbance paper or paper towel

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Androstenedione R3 RC

STORAGE AND STABILITY

1. Store the kit at 2 - 8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose reagent to heat, sun, or strong light.

WARNINGS AND PRECAUTIONS

The Standard contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.

2. This test kit is designed for research use only.

3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled. 4. The components in this kit are intended for use as an integral unit. The components of different lots should not

be mixed.

5. It is recommended that standards, control and serum samples be run in duplicate.

6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.

SPECIMEN COLLECTION HANDLING

1. Collect blood specimens and separate the serum immediately.

2. Specimens may be stored refrigerated at (2-8° C) for 1 week. If storage time exceeds 1 week, store frozen at (-20° C) for up to one month.

3. Avoid multiple freeze-thaw cycles.

4. Prior to assay, frozen sera should be completely thawed and mixed well. 5. Do not use grossly lipemic specimens.

PREPARATION OF REAGENTS

20XWash Buffer: Prepare 1X Wash Buffer by adding the contents of the bottle (25ml, 20X) to 475 ml of distilled or

deionized water. Store at room temperature (18-26° C).

ASSAY PROCEDURE

All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption.

1. Secure the desired number of microwells strips in the holder.

2. Dispense 25µl Androstenedione Standards, controls and samples into appropriate wells. 3. Dispense 50µl anti- Androstenedione reagent into each well.

4. Dispense 50µl Enzyme Conjugate into each well. 5. Incubate for 60 minutes at room temperature with shaking.

6. Briskly shake out the contents of the wells. Rinse the wells 3 times with diluted wash solution. Strike the wells sharply on absorbent paper to remove residual water droplets.

NOTE: The sensitivity and precision of this assay is markedly influenced by the correct performance of the

washing procedure.

7. Add 100 µl of Substrate Solution to each well. 8. Incubate for 15 minutes at room temperature.

9. Stop the enzymatic reaction by adding 50µl of Stop Solution into each well.

10. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stop solution.

1. Calculate the average absorbance values for each set of standards, controls and patient samples

2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration in ng/ml with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis

3. Using the mean absorbance value for each sample determine the corresponding concentration of Androstenedione from the standard curve. Depending on experience and/or the availability of computer capability, other methods of data reduction may be employed.

4. Automated method: Computer programs using cubic spline, 4 PL (4 Parameter Logistics) or Logit-Log can generally give a good fit.

5. The concentration of the samples can be read directly from this standard curve. Samples with Androstenedione concentration higher than the concentration of the highest standard have to be diluted with zero standard. For the calculation of the concentrations this dilution factor has to be taken into account.

Example of a standard Curve

OD 450 nm Conc. ng/mL Std 1 2.132 0 Std 2 1.705 0.12 Std 3 1.324 0.37 Std 4 0.811 1.11 Std 5 0.314 3.33 Std 6 0.171 10 EXPECTED VALUES

It is recommended that each laboratory establish its own normal ranges based on a representative sampling of the local population. The following values may be used as initial guideline ranges only:

Age Conc. Range ng/ml

Male and Pre-Menopausal women Adult 0.25 – 3.0 Post-menopausal women Adult 0.12 – 1.5

LIMITATION OF THE TEST

1. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.

PERFORMANCE CHARACTERISTICS

1. Sensitivity

The sensitivity was determined by calculating the mean plus 2SD of the standard zero point tested 20 times in the same run.

Serum _ReplicatesNo. of Mean _ng/ml Standard _Deviation Mean + 2SD (Sensitivity) ng/ml Zero

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AFP R1RC REFERENCES

1. Bates SE. Clinical applications of serum tumor markers. Ann Intern Med 1991;115:623-8.

2. Wu JC, Lee SD, Hsaio KJ, et al. Mass screening of primary hepatocellular carcinoma by alpha-fetoprotein in a rural area of Taiwan-a dried blood spot method. Liver 1988;8:100-4.

3. Lee H-S, Chung YH, Kim CY. Specificities of serum alpha-fetoprotein in HBsAg+ and HBsAg- patients in the diagnosis of hepatocellular carcinoma. Hepatology 1991;14:68-72.

4. Di Bisceglie AM, Rustgi VK, Hoofnagle JH, Dusheiko GM, Lotze MT. Hepatocellular carcinoma. Ann Intern Med 1988;108:390-401.

5. Sato Y, Nakata K, Kato Y, et al. Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein. N Engl J Med 1993;328:1802-6.

6. Deutch HF. Chemistry and biology of alpha- fetoprotein. Adv Cancer Res 1991;56:253-312. 2014-07-23

Cat#: AF237T (96 Tests) For Order and Inquiries, please contact

Calbiotech Inc.,

www.calbiotech.com

Alpha-Fetoprotein (AFP) ELISA

Catalog No. AF237T (96 tests)

INTENDED USE

The Calbiotech AFP ELISA Kit is intended for the quantitative measurement of AFP in human serum. For research use only.

Alpha fetoprotein (AFP) is a glycoprotein with a molecular weigh of approximately 70,000 Daltons. AFP is normally produced during fetal and neonatal development by the liver, yolksac, and in small concentrations by the gastrointestinal tract. After birth, serum AFP concentrations decrease rapidly, and by the second year of life and thereafter only trace amounts are normally detected in serum.

Elevation of serum AFP to abnormally high values occurs in several malignant diseases, most notably nonseminomatous testicular cancer and primary hepatocellular carcinoma. In the case of nonseminomatous testicular cancer, a direct relationship has been observed between the incidence of elevated AFP levels and the stage of disease. Elevated AFP levels have also been observed in patients diagnosed with seminoma with nonseminomatous elements, but not in patients with pure seminoma. In addition, elevated serum AFP concentrations have been measured in patients with other noncancerous diseases, including ataxia telangiectasia, hereditary tyrosinemia, neonatal hyperbilirubinemia, acute viral hepatitis, chronic active hepatitis, and cirrhosis. Elevated serum AFP concentrations are also observed in pregnant women. Therefore, AFP measurements are not recommended for use as a screening procedure to detect the presence of cancer in the general population.

This AFP ELISA kit is a solid phase sandwich assay method, based on a streptavidin-biotin principle. The standards, samples and the biotinylated Anti-AFP antibody reagent are added into designated wells, coated with Streptavidin. Endogenous AFP in the patient’s serum binds to the antigenic site of the biotinylated Anti-AFP antibody. Simultaneously, the biotinylated antibody is immobilized onto the wells through the high affinity Streptavidin-Biotin interaction. Unbound protein and excess biotin conjugated antibody are washed off by wash buffer. Upon the addition of the Peroxidase (HRP) conjugated Anti-AFP antibody reagent, a sandwich complex is formed, the analyte of interest being in between the two highly specific antibodies, labeled with Biotin and HRP. Unbound protein excess enzyme conjugated antibody reagent is washed off by wash buffer. Upon the addition of the substrate, the intensity of color developed is directly proportional to the concentration of AFP in the samples. A standard curve is prepared relating color intensity to the concentration of the AFP.

MATERIALS PROVIDED 96 Tests 1. Microwell coated with Streptavidin 12x8x1 2. AFP Standard: 6 vials (ready to use) 0.5ml 3. Anti-AFP Enzyme Conjugate: 1 bottle (ready to use) 12ml 4. Anti-AFP-Biotin Reagent: 1 bottle (ready to use) 12ml 5. TMB Substrate: 1 bottle (ready to use) 12ml 6. Stop Solution: 1 bottle (ready to use) 12ml 7. 20X Wash concentrate: 1 bottle 25ml

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AFP R1RC MATERIALS NOT PROVIDED

4. ELISA reader capable of reading absorbance at 450nm 5. Absorbance paper or paper towel

6. Graph paper

STORAGE AND STABILITY 1. Store the kit at 2 - 8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose test reagents to heat, sun, or strong light. WARNINGS AND PRECAUTIONS

The calibrator and controls contain human source components which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984

2. This kit is for research use only.

5. It is recommended that serum samples be run in duplicate.

6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.

SPECIMEN COLLECTION HANDLING

1. Collect blood specimens and separate the serum immediately.

2. Specimens may be stored refrigerated at (2-8°C) for 5 days. If storage time exceeds 5 days, store frozen at (-20° C) for up to one month.

3. Avoid multiple freeze-thaw cycles.

4. Prior to assay, frozen sera should be completely thawed and mixed well. 5. Do not use grossly lipemic specimens.

REAGENTS PREPARATION

Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-26° C).

ASSAY PROCEDURE

Prior to assay, allow reagents to stand at room temperature. Gently mix all reagents before use. 1. Place the desired number of coated strips into the holder

2. Pipette 25 µl of AFP standards, control and patient’s sera.

3. Add 100 µl of Anti-AFP-Biotin Reagent to all wells and mix for 20-30 seconds. 4. Cover the plate and incubate for 30 minutes at room temperature (18-26° C).

5. Remove liquid from all wells. Wash wells three times 300 µl with 1X wash buffer. Blot on absorbent paper towels.

6. Add 100 µl of the Anti-AFP- Enzyme conjugate to all wells. Cover and incubate for 30 minutes. 7. Remove liquid from all wells. Wash wells three times 300 µl with 1X wash buffer. Blot on absorbent

paper towels.

8. Add 100 µl of TMB substrate to all wells. 9. Incubate for 15 minutes at room temperature.

10. Add 50 µl of stop solution to all wells. Shake the plate gently to mix the solution.

11. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution. CALCULATION OF RESULTS

The standard curve is constructed as follows:

1. Check AFP standard value on each standard vial. This value might vary from lot to lot. Make sure you check the value on every kit. See example of the standard attached.

2. To construct the standard curve, plot the absorbance for the AFP standards (vertical axis) versus the AFP standard concentrations in ng/ml (horizontal axis) on a linear graph paper. Draw the best curve through the points.

3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control or unknown sample.

Example of a Standard Data

OD 450 nm Conc. ng/mL Std 1 0.020 0 Std 2 0.072 5 Std 3 0.281 25 Std 4 0.462 50 Std 5 1.878 250 Std 6 2.447 500

LIMITATIONS OF THE TEST

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ANA ELISA R3 RC REFERENCES

1. Emlen W; O'Neill L Clinical significance of antinuclear antibodies: comparison of detection with immunofluorescence and enzyme-linked immunosorbent assays. Arthritis Rheum 1997;40(9):1612-8.

2. González C; Martin T; Arroyo T; García-Isidoro M; Navajo JA; González-Buitrago JM. Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA). J Clin Lab Anal 1997;11(6):388-92.

3. Parveen S; Morshed SA; Nishioka M. High prevalence of antibodies to recombinant CENP-B in primary biliary cirrhosis: nuclear immunofluorescence patterns and ELISA reactivities. J Gastroenterol Hepatol 1995;10(4):438-45.

4. Welin Henriksson E; Hansson H; Karlsson-Parra A; Pettersson I. Autoantibody profiles in canine ANA-positive sera investigated by immunoblot and ELISA. Vet Immunol

Immunopathol 1998;61(2-4):157-70.

5. Koh WH; Dunphy J; Whyte J; Dixey J; McHugh NJ. Characterisation of anticytoplasmic antibodies and their clinical associations [see comments]. Ann Rheum Dis

1995;54(4):269-73.

6. Spronk PE; Bootsma H; Horst G; Huitema MG; Limburg PC; Cohen Tervaert JW; Kallenberg CG. Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus. Br J Rheumatol 1996;35(7):625-31.

2014-07-07

Cat#: AN033G (96 Tests) For Order and Inquiries, please contact

Calbiotech Inc.,

www.calbiotech.com

ANA Screen ELISA

Catalog No.: AN033G (96 Tests) INTENDED USE

The Calbiotech Inc. (CBI) ANA Screen ELISA test system is an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG class antibodies to ANA in human serum or plasma.For research use only.

Antinuclear antibodies (ANA) are frequently present in patients with systemic lupus erythematosus (SLE) and, less commonly, in other autoimmune diseases Rheumatoid arthritis, Collagen vascular diseases, chronic liver diseases and systemic sclerosis (scleroderma). ANA bind to several nuclear antigens including DsDNA, SSDNA, RNP, Sm, SSA and SSB. ANA frequency increases with age in apparently healthy people, especially women after the age of 45 years. ANA ELISA is widely used as a screening procedure for different autoimmune diseases.

Diluted patient serum is added to wells coated with purified nuclear antigens. ANA IgG specific antibody, if present, binds to the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. The plate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the color generated is proportional to the amount of IgG specific antibody in the sample.

MATERIALS PROVIDED 96 Tests

1. Microwells coated with nuclear antigens 12x8x1

2. Sample Diluent: 1 bottle (ready to use) 22 ml

3. Calibrator 1 Vial (ready to use) 1ml

4. Positive Control 1 vial (ready to use) 1ml

5. Negative Control 1 vial (ready to use) 1ml

6. Enzyme conjugate: 1 bottle (ready to use) 12ml

7. TMB Substrate: 1 bottle (ready to use) 12ml

8. Stop Solution: 1 bottle (ready to use) 12ml

9. Wash concentrate 20X: 1 bottle 25ml

MATERIALS NOT PROVIDED 1. Distilled or deionized water. 2. Precision pipettes. 3. Disposable pipette tips.

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ANA ELISA R3 RC 5. Absorbance paper or paper towel.

STORAGE AND STABILITY 1. Store the kit at 2 – 8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose test reagents to heat, sun or strong light. WARNINGS AND PRECAUTIONS

The calibrator and controls contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984

2. This kit is designed for research use only.

6. This product contains components preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.

SPECIMEN COLLECTION AND HANDLING

1. Collect blood specimens and separate the serum.

2. Specimens may be refrigerated at 2–8 °C for up to seven days or frozen for up to six months. Avoid repetitive freezing and thawing.

REAGENT PREPARATION

Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-26 °C).

PREPARATION FOR ASSAY

Bring all specimens and kit reagents to room temperature (18-26 °C) and gently mix. ASSAY PROCEDURE

1. Place the desired number of coated strips into the holder.

2. Negative control, positive control, and calibrator are ready to use. Prepare 1:21 dilution of test samples, by adding 10 µl of the sample to 200 µl of sample diluent. Mix well. 3. Dispense 100 µl of diluted sera, calibrator and controls into the appropriate wells. For

the reagent blank, dispense 100µl sample diluent in 1A well position. Tap the holder to

remove air bubbles from the liquid and mix well. Incubate for 20 minutes at room temperature.

4. Remove liquid from all wells. Wash wells three times with 300 µL of 1X wash buffer. Blot on absorbance paper or paper towel.

5. Dispense 100 µl of enzyme conjugate to each well and incubate for 20 minutes at room temperature.

6. Remove enzyme conjugate from all wells. Wash wells three times with 300 µl of 1X wash buffer. Blot on absorbance paper or paper towel

7. Dispense 100 µl of TMB substrate and incubate for 10 minutes at room temperature. 8. Add 100 µl of stop solution.

9. Read O.D. at 450 nm using ELISA reader within 15 min. A dual wavelength is recommended with reference filter of 600-650 nm.

1. Check Calibrator Factor (CF) value on the calibrator bottle. This value might vary from lot to lot. Make sure you check the value on every kit.

2. Calculate the cut-off value: Calibrator OD x Calibrator Factor (CF).

3. Calculate the Ab (Antibody) Index of each determination by dividing the O.D. value of each sample by cut-off value.

Example of typical results: Calibrator mean OD = 0.8 Calibrator Factor (CF) = 0.5

Cut-off Value = 0.8 x 0.5= 0.400 Positive control O.D. = 1.2 Ab Index = 1.2 / 0.4 = 3

Patient sample O.D. = 1.6 Ab Index = 1.6 / 0.4 = 4.0

QUALITY CONTROL

The test run may be considered valid provided the following criteria are met: 1. If the O.D. of the Calibrator should be greater than 0.250.

2. The Ab index for Negative control should be less than 0.9. 3. The Ab index for Positive control should be greater than 1.2. INTERPRETATION

The following is intended as a guide to interpretation of ANA IgG test results; each laboratory is encouraged to establish its own criteria for test interpretation based on sample populations encountered.

Antibody Index Interpretation

<0.9 No detectable ANA IgG by ELISA.

0.9-1.1 Borderline positive. Follow-up testing is recommended if clinically indicated. >1.1 Detectable ANA IgG by ELISA.

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Brucella IgG R4 RC 2. Precision

Intra Assay Study

Serum _ReplicatesNo. of Mean _DeviationStandard Coefficient of _{Variation %}

1 16 1.31 0.071 5.41

2 16 0.86 0.052 6.04

3 16 0.24 0.015 6.25

Inter Assay Study

1 10 1.92 0.21 10.93

2 10 1.44 0.17 11.80

3 10 0.25 0.032 12.80

REFERENCES

1. Gad El-Rab MO; Kambal AM. Evaluation of a Brucella enzyme immunoassay test (ELISA) in comparison with bacteriological culture and agglutination. J Infect 1998; 36(2):197-201.

2. Mikolon AB; Gardner IA; Hietala SK; Hernandez de Anda J; Chamizo Pestana E; Hennager SG; Edmondson AJ. Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats. J Clin Microbiol 1998; 36(6):1716-22.

3. Bowden RA; Cloeckaert A; Zygmunt MS; Bernard S; Dubray G. Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry. Infect Immun 1995; 63(10):3945-52.

4. Baldi PC; Miguel SE; Fossati CA; Wallach JC. Serological follow-up of human brucellosis by measuring IgG antibodies to lipopolysaccharide and cytoplasmic proteins of Brucella species. Clin Infect Dis 1996;22(3):446-55 5. Casao MA; Leiva J; Diaz R; Gamazo C. Anti-phosphatidylcholine antibodies in patients with brucellosis. J Med

Microbiol 1998; 47(1):49-54.

2014-07-07

Cat#: BA052G (96 Tests) For Order and Inquiries, please contact

Brucella IgG ELISA

Catalog No. BA052G (96 Tests) INTENDED USE

The Calbiotech Brucella IgG ELISA Kit is intended for the detection of IgG antibody to Brucella in human serum or plasma. For research use only.

Brucella is a gram negative coccobacilli capable of infecting a wide range of animal and man. Of the three species causing human infection, B. melitensis is the most patogenic followed by B. suis and B. abortus . Brucellosis is transmitted through contaminated and untreated milk and milk products and by direct contact with infected animals (cattle, sheep, goats, pigs, camels, buffaloes, and, very recently, seals), animal carcasses, and abortion materials. Worldwide, millions of individual are at risk, especially in developing countries where the infection in animals has not been brought under control, heat treatment procedures of milk (e.g. pasteurization) are not routinely applied, and food habits such as consumption of raw milk. The incubation period of brucellosis is usually one to three weeks, but sometimes may be several months. The illness may be mild and self-limiting or severe. The disease is accompanied by continued, intermittent, or irregular fever, headache, weight loss and generalized aching and fatigue. Urogenital symptoms may dominate the clinical presentation in some patients.

This method uses B. abortus outer membrane, which is shared by the other species. Brucella IgG and IgA antibodies persist for many years after infection. A significant increase in Brucella IgG level is in patients with symptoms of brucellosis is indicative of recent exposure. IgM antibodies are present in acute brucellosis and also found in about 33% of patients with chronic brucellosis.

Diluted patient serum is added to wells coated with purified antigen. IgG specific antibody, if present, binds to the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. The plate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the color generated is proportional to the amount of IgG specific antibody in the sample.

MATERIALS PROVIDED 96 Tests 1. Microwell coated with Brucella abortus antigen 12x8x1 2. Sample Diluent: 1 bottle (ready to use) 22 ml 3. Calibrator: 1 Vial (ready to use) 1ml 4. Positive Control: 1 vial (ready to use) 1ml 5. Negative Control: 1 vial (ready to use) 1ml 6. Enzyme conjugate: 1 bottle (ready to use) 12ml 7. TMB Substrate: 1 bottle (ready to use) 12ml 8. Stop Solution: 1 bottle (ready to use) 12ml 9. Wash concentrate 20X: 1 bottle 25ml MATERIALS NOT PROVIDED

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Brucella IgG R4 RC 4. ELISA reader capable of reading absorbance at 450nm

5. Absorbance paper or paper towel 6. Graph paper

STORAGE AND STABILITY 1. Store the kit at 2-8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose test reagents to heat, sun or strong light. WARNINGS AND PRECAUTIONS

The calibrator and controls contain human source components which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984.

2. This kit is for research use only. Not for use in diagnostic procedures.

6. Control sera and sample diluent contain preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.

SPECIMEN COLLECTION AND HANDLING 1. Collect blood specimens and separate the serum.

ASSAY PROCEDURE

Prior to assay, allow reagents ti stand at room temperature. Gently mix all reagents before use. 1. Bring all specimens and kit reagents to room temperature (18-26 °C) and gently mix. 2. Place the desired number of coated strips into the holder.

3. Negative control, positive control, and calibrator are ready to use. Prepare 1:21 dilution of test samples, by adding 10 µl f the sample to 200 µl f sample diluent. Mix well.

4. Dispense 100 µl f diluted sera, calibrator and controls into the appropriate wells. For the reagent blank, dispense 100µl ample diluent in 1A well position. Tap the holder to remove air bubbles from the liquid and mix well. Incubate for 20 minutes at room temperature.

5. Remove liquid from all wells. Wash wells three times with 300µl of 1X wash buffer. Blot on absorbance paper or paper towel.

6. Dispense 100 µl of enzyme conjugate to each well and incubate for 20 minutes at room temperature. 7. Remove enzyme conjugate from all wells. Wash wells three times with 300 µl of 1X wash buffer. Blot

on absorbance paper or paper towel.

Example of typical results:

Calibrator mean OD = 0.8 Calibrator Factor (CF) = 0.5 Cut-off Value = 0.8 x 0.5= 0.400 Positive control O.D. = 1.2 Ab Index = 1.2 / 0.4 = 3

QUALITY CONTROL

The test run may be considered valid provided the following criteria are met: 1. The O.D. of the Calibrator should be greater than 0.250.

2. The Ab index for Negative control should be less than 0.9. 3. The Ab Index for Positive control should be greater than 1.2.

INTERPRETATION

The following is intended as a guide to interpretation of Brucella IgG test results; each laboratory is encouraged to establish its own criteria for test interpretation based on sample populations encountered.

<0.9 No detectable antibody to Brucella IgG by ELISA.

0.9-1.1 Borderline positive. Follow-up testing is recommended if clinically indicated. >1.1 Detectable antibody to Brucella IgG by ELISA.

1. Lipemic or hemolyzed samples may cause erroneous results.

1. Sensitivity and Specificity

92 patient sera were tested by this Brucella IgG ELISA and a reference ELISA method. 14 sera were positive and 77 were negative by both methods (99% agreement). The results are summarized below:

Brucella IgG ELISA

+ − Total

Reference ELISA Kit + 14 0 14 _ 1 77 78

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Brucella IgM Rev.4RC 2. Precision

Intra Assay Study

1 16 1.49 0.066 4.43

2 16 1.01 0.051 5.50

3 16 0.19 0.012 6.31

Inter Assay Study

1 10 1.41 0.139 09.85

2 10 0.97 0.100 10.30

3 10 0.20 0.022 11.00

REFERENCES

1. Gad El-Rab MO; Kambal AM. Evaluation of a Brucella enzyme immunoassay test (ELISA) in comparison with bacteriological culture and agglutination. J Infect 1998; 36(2):197-201.

2. Mikolon AB; Gardner IA; Hietala SK; Hernandez de Anda J; Chamizo Pestana E; Hennager SG; Edmondson AJ. Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats. J Clin Microbiol 1998; 36(6):1716-22.

3. Bowden RA; Cloeckaert A; Zygmunt MS; Bernard S; Dubray G. Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry. Infect Immun 1995; 63(10):3945-52.

4. Baldi PC; Miguel SE; Fossati CA; Wallach JC. Serological follow-up of human brucellosis by measuring IgG antibodies to lipopolysaccharide and cytoplasmic proteins of Brucella species. Clin Infect Dis 1996;22(3):446-55 5. Casao MA; Leiva J; Diaz R; Gamazo C. Anti-phosphatidylcholine antibodies in patients with brucellosis. J Med

Microbiol 1998; 47(1):49-54.

2014-07-07

Cat#: BA053M (96 Tests) For Order and Inquiries, please contact

Brucella IgM ELISA

Catalog No.: BA053M (96 Tests) INTENDED USE

The Calbiotech, Inc (CBI) Brucella IgM ELISA Kit is intended for the detection of IgM antibody to Brucella in human serum or plasma. For research use only.

Brucella is a gram negative coccobacilli capable of infecting a wide range of animal and man. Of the three species causing human infection, B. melitensis is the most patogenic followed by B. suis and B. abortus . Brucellosis is transmitted through contaminated and untreated milk and milk products and by direct contact with infected animals (cattle, sheep, goats, pigs, camels, buffaloes, and, very recently, seals), animal carcasses, and abortion materials. Worldwide, millions of individual are at risk, especially in developing countries where the infection in animals has not been brought under control, heat treatment procedures of milk (e.g. pasteurization) are not routinely applied, and food habits such as consumption of raw milk. The incubation period of brucellosis is usually one to three weeks, but sometimes may be several months. The illness may be mild and self-limiting or severe. The disease is accompanied by continued, intermittent, or irregular fever, headache, weight loss and generalized aching and fatigue. Urogenital symptoms may dominate the clinical presentation in some patients.

This method uses B. abortus outer membrane, which is shared by the other species. Brucella IgG and IgA antibodies persist for many years after infection. A significant increase in Brucella IgG level is in patients with symptoms of brucellosis is indicative of recent exposure. IgM antibodies are present in acute brucellosis and also found in about 33% of patients with chronic brucellosis.

Diluted patient serum (serum diluent contains sorbent to remove Rheumatoid Factor and human IgG interference) is added to wells coated with purified antigen. IgM specific antibody, if present, binds to the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. The plate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the color generated is proportional to the amount of IgM specific antibody in the sample.

MATERIALS PROVIDED 96 Tests 1. Microwell coated with Brucella antigen 12x8x1 2. Sample Diluent: 1 bottle (ready to use) 22 ml 3. Calibrator: 1 Vial (ready to use) 1ml 4. Positive Control: 1 vial (ready to use) 1ml 5. Negative Control: 1 vial (ready to use) 1ml 6. Enzyme conjugate: 1 bottle (ready to use) 12ml 7. TMB Substrate: 1 bottle (ready to use) 12ml 8. Stop Solution: 1 bottle (ready to use) 12ml 9. Wash concentrate 20X: 1 bottle 25ml MATERIALS NOT PROVIDED

(14)

Brucella IgM Rev.4RC 4. ELISA reader capable of reading absorbance at 450nm

5. Absorbance paper or paper towel3 6. Graph paper

STORAGE AND STABILITY

1. Store the kit at 2-8° C.

2. Keep microwells sealed in a dry bag with desiccants. 3. The reagents are stable until expiration of the kit. 4. Do not expose test reagents to heat, sun or strong light.

WARNINGS AND PRECAUTIONS

The calibrator and controls contain human source components which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984.

2. This kit is for research use only.

6. Control sera and sample diluent contain preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.

SPECIMEN COLLECTION AND HANDLING

1. Collect blood specimens and separate the serum.

ASSAY PROCEDURE

Bring all specimens and kit reagents to room temperature (18-26 °C) and gently mix. 1. Place the desired number of coated strips into the holder.

2. Negative control, positive control, and calibrator are ready to use. Prepare 1:21 dilution of test samples, by adding 10 µl of the sample to 200 µl of sample diluent. Mix well.

3. Dispense 100 µl of diluted sera, calibrator and controls into the appropriate wells. For the reagent blank, dispense 100µl sample diluent in 1A well position. Tap the holder to remove air bubbles from the liquid and mix well. Incubate for 20 minutes at room temperature.

4. Remove liquid from all wells. Wash wells three times with 300µl of 1X wash buffer. Blot on absorbance paper or paper towel.

5. Dispense 100 µl of enzyme conjugate to each well and incubate for 20 minutes at room temperature. 6. Remove enzyme conjugate from all wells. Wash wells three times with 300µl of 1X wash buffer. Blot on

absorbance paper or paper towel.

Example of typical results:

Calibrator mean OD = 0.8 Calibrator Factor (CF) = 0.5 Cut-off Value = 0.8 x 0.5= 0.400 Positive control O.D. = 1.2 Ab Index = 1.2 / 0.4 = 3

QUALITY CONTROL

The test run may be considered valid provided the following criteria are met: 1. The O.D. of the Calibrator should be greater than 0.250.

2. The Ab index for Negative control should be less than 0.9. 3. The Ab Index for Positive control should be greater than 1.2.

INTERPRETATION

The following is intended as a guide to interpretation of Brucella IgM test results; each laboratory is encouraged to establish its own criteria for test interpretation based on sample populations encountered.

<0.9 No detectable antibody to Brucella IgM by ELISA.

0.9-1.1 Borderline positive. Follow-up testing is recommend if clinically indicated. >1.1 Detectable antibody to Brucella IgM by ELISA.

1. To enhance sensitivity and specificity of this IgM test provided sample diluent has been formulated to block IgG and Rheumatoid Factor (RF) interferences. Turbidity could be seen after diluting serum with sample diluent. This turbidity is due to the blocking of serum IgG and shows no interference with test results. It can be removed by centrifugation.

2. In specimens with high RF and high autoimmune antibodies, the possibility of eliminating the interferences cannot be ruled out entirely.

3. Lipemic or hemolyzed samples may cause erroneous results.

1. Sensitivity and Specificity

178 patient sera were tested by this Brucella IgM ELISA and a reference ELISA method. 26 sera were positive and 149 were negative by both methods (98% agreement). The results are summarized below:

Brucella IgM ELISA

+ − Total

Reference ELISA Kit + 23 2 25 _ 1 152 153 Total 24 154 178