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Recibido en noviembre de 1995. Aceptado en marzo de 1996. A new UMELISA format for the quantification of maternal serum alpha-fetoprotein Ivonne Gomez Cordero, Juliette Cazanave Mora, Rosa Lidia Solis Rodriguez, Celina Machado Rodriguez, Dunia Becquer Ariza and Jose Luis Fernandez Yero Immunoassay Center (CIE), P.O. Box 6945, Havana 6 C.P. 10600, Cuba.
Code Number: BA96042
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Abstract In our country, the quantification of Alpha-fetoprotein is performed using a sandwich type heterogeneous enzyme immunoassay (UMELISA-AFP, 10 uL of samples and reagents) compatible with SUMA (Ultramicroanalytical system) technology designed mainly for screening purposes. This Kit has been modified, for improved manipulation and use. This paper shows standardization results of this new format (ready to use), its comparison with Enzymun- Test AFP of Boehringer Mannheim (Code 1361236) and screening results in our country. Stability is always higher than 80% in the new presentation of the reagents that form the Kit (after 18 months). In the linear regression analysis performed between the new UMELISA format and the former one, a correlation of 0.9998, a slope of 1.07 and an angle of 44 degrees, were obtained. In the comparison between the International Standard 72/225 of The World Health Organization (WHO) and our standard, we obtained a correlation of 0.9996, a slope of 0.9798 and an angle of 44 degrees. Variation coefficients intra and inter-assay, obtained in the accuracy assay were less than 5%. A correlation of 0.9380 was given by the correlation assay with 36 samples with the Enzymun-Test AFP of Boehringer Mannheim. Key words: Alpha-fetoprotein, new UMELISA format, SUMA Resumen En nuestro pais la cuantificacion de Alfa-fetoproteina se realiza a traves de un ensayo inmunoenzimatico heterogeneo de tipo sandwich (UMELISA-AFP, 10 uL de muestras y reactivos), compatible con la tecnologia SUMA (Sistema Ultramicroanalitico), designada fundamentalmente para propositos de pesquisaje. Este estuche diagnostico ha sido modificado con la finalidad de lograr una mejor utilizacion y manipulacion del mismo. En el presente trabajo se reflejan los resultados de la estandarizacion de este nuevo formato (listo para el uso), su comparacion con el Enzymun- Test AFP de la Boehringer Mannheim (Codigo 1361236) y los resultados en el pesquisaje realizado en nuestro pais. La estabilidad obtenida en la nueva presentacion de los reactivos que conforman el estuche siempre fue superior al 80%, despues de 18 meses. En el analisis de regresion linear realizado entre el nuevo formato y el de uso, se obtuvo una correlacion de 0,9998, una pendiente de 1,07, y un angulo de 44. Al comparar nuestro estandar con el Estandar Internacional 72/225 de la Organizacion Mundial de la Salud (OMS) se obtuvo una correlacion de 0,9996, una pendiente de 0,9798 y un angulo de 44. En el ensayo de precision se obtuvieron coeficientes de variacion intra e inter- ensayo menores del 5%. La correlacion con 36 muestras con el Enzymun-Test AFP de la Boehringer Mannheim dio como resultado una correlacion de 0,9380. Palabras claves: Alfafetoproteina, nuevo formato UMELISA, SUMA Introduction Alpha-fetoprotein (AFP) is a glycoprotein of approximately 70 000 Da, usually found in normal serum, in minimal concentrations which increase physiologically during pregnancy (1-3). Its quantification, in maternal serum, is relevant for the screening of neural tube defects (NTD), open defects in the anterior ventral wall, twin pregnancy, threats of abortion, fetal death and others (4-8). The low AFP maternal serum values are associated to chromosome abnormalities in the fetus, such as Down's Syndrome (9,10). It is a marker associated to hepatocellular carcinomas and tumors of the ovary and testicle germinal cells. Serum AFP also increases in the presence of other malignant tumors such as; gastric and pancreatic carcinomas, cholangiocarcinomas, esophageal carcinomas and non neoplastic hepatic diseases (hepatitis and cirrhosis). The Ultramicroanalytical system (SUMA) was developed in our center in the last decade. It was designed mainly for screening purposes. This includes SUMA equipment and reagents used in the UltramicroELISA test (UMELISA), which combines high sensitivity of current ELISA tests with the use of ultramicrovolumes of samples and reagents (10 uL or less). Immunoassays are highly related with quantification of Alpha -fetoprotein in maternal serum (11-14). A sandwich type heterogeneous enzyme immunoassay for the quantification of AFP in pregnant women between 15 and 19 weeks of pregnancy for pre-natal diagnostic (15) was introduced in our country in 1982. It was compatible with the SUMA technology, making the assay specific, accurate, rapid, of low cost and applicable to a large number of samples at the same time and very useful in screening protocols (16-19). The useful life time of the Kit format was affected by the low stability of the reagents, mainly the fluorogenic substrate (4-methylumbelliferyl phosphate) and that is the reason why a new one has been developed. Another limitation was the solid phase used (irradiated polystyrene plates with a capacity of 39 duplicated determinations). If the amount of samples to process were less than this number, the rest of the plate positions could be wasted or results would be delayed on awaiting to complete this number. Materials and Methods Solid Phase Irradiated polystyrene plates (8 X 12 strips) and with titanium dioxide (Greinerlabortechnik, Germany) were used as a solid phase (20). Antibodies Polyclonal antibodies to AFP were obtained from the Immunoassay Center. Amniotic fluid with raised level of AFP (in a concentration higher than 200 ug/mL, quantified by electroimmunodiffusion) was used as an antigen. Antigen was purified by ionic exchange chromatography in DEAE Sephadex matrix and a K26/40 ion-exchange column in order to attain a high purity level. Afterwards, antigen was purified by affinity chromatography (AH-Sepharosa 4B-anti AFP) and it was dialized in Sephadex G-25 Coarse. Then, sheep were immunized using this antigen. The antibodies obtained, were purified by affinity chromatography AH-Sepharose 4B glutaraldehyde-AFP and eluted with a 0.2 M glycine buffer, pH 2.8. They were desalted versus phosphate-buffered saline, pH 7.4 (PBS), and stored in the same buffer at -20 øC until their use. Antibody-coated Solid Phase The solid phase used was coated with 15 uL of polyclonal AFP antibodies at a concentration of 8 ug/mL in 0.1 M carbonate-bicarbonate buffer, pH 9.6; the plates were incubated during 4 hours in a humidity chamber at 45 øC, and then they were blocked with PBS containing 0.1% bovine serum albumin (BSA) and sealed. Washing buffer Tris (hydroximethyl)methylamine-HCl, 0.015 M pH 7.8, (BDH Chemicals Ltd. Poole, England). Preparation of standard sera, control serum and samples The standard sera and control serum were prepared in CIE from normal human serum with amniotic fluid (Maternal Hospitals and from Blood Banks, in Havana City), and they were negative to anti-HIV 12, HBsAg and Anti-HCV. The standard serum was presented in a lyophilized form at a concentration of 100 IU/mL; the new standard serum is presented in the form of independent concentrations which are: 96 IU/mL, 48 IU/mL, 24 IU/mL, 12 IU/mL, 6 IU/mL and 0 IU/mL (this has only Human Serum). In the reconstitution of the Standard Sera and the Control Serum and in the dilution of samples, a sheep serum was used in two different forms: at 20% and without dilution; both were reconstituted with Tris, obtaining a final concentration of 5%. The dilution of samples was 1:2 in both solutions. Antibody-enzyme Conjugate A polyclonal anti-AFP antibody was conjugated with alkaline phosphatase EIA-grade I (Boehringer Mannheim GmbH, Germany), using the one-step glutaraldehyde method developed by Avrameas (21). This conjugate was used in a concentrated and ready to use solution (diluted in Tris pH 8.0 and BSA at 1%). Substrate preparation The 4-methylumbelliferyl phosphate substrate (4-MUP) (Koch Light Ltd. Haverhill, Suffolk, England), was prepared in two different forms: a lyophilized form and a 0.5 mM concentrated solution. Diethanolamine buffer 0.92 M pH 9.8 (Merck, Schuchardt, for synthesis) was used for its dilution. As a reference solution, 0.057 mM 4-methylumbelliferone was used. Equipment-SUMA fluorimeter 421 To measure the intensity of the emitted fluorescence due to the enzymatic activity and the non-enzymatic hydrolysis a computerized fluorimeter (SUMA) was used. The SUMA has a high sensitivity and low operation cost; it reads fluorescence in less than 1 minute. The reading is performed between the 420 and 540 nm wavelengths. ERIZO Multipipette 201 It is used to manipulate liquids and to prepare samples in immunotesting techniques that require great precision, in a volume interval between 2 and 200 uL. MAS Washer 201 It is a fully-automatic device used to guarantee uniformity in the washing procedures of the UltramicroELISA plates. The accessories of SUMA laboratory and the software package UMELISA-AFP were also used. Determination of residual humidity using Karl Fisher's method Determination is performed as a dead-stop endpoint (DSEP) titration using a double platinum electrode, with the Radiometer TTT 83KF-TITRATOR (Copenhagen, Denmark). The potentiometric titration is based on the reaction between the iodine methanolic solution and water in the presence of sulphur dioxide and pyridine. Chemical reactions abruptly change the resistance and these sudden changes, detected by the introduction of a double platinum electrode in the solution, indicate that the equivalence point has been reached. Test Procedures Samples are incubated in strip wells during 1 hour at 37 C. Unbound elements are removed after three washings with Tris buffer and only the antibody/AFP complex remains in the wells. After washing them, 10 uL of alkaline phosphatase (A.P.)-conjugate anti AFP were added and incubated 1 hour at 37 øC. Strips are washed again to eliminate the excess of the conjugate. Then the wells were incubated for 30 minutes at room temperature with 10 uL of 4-MUP that is hydrolyzed by the enzyme of the conjugate. The fluorescence intensity will be proportional to the AFP concentration in the sample. Statistical analysis The statistical parameters were obtained using the appropriate tests in the StatGraphic software on an IBM-compatible computer. Precision Analyte concentration was measured repeatedly in three human sera that contained AFP in three ranges of values: high, medium and low. Sensitivity The lowest limit of detection (mean +2 SD of results for zero standard) was calculated from the results of assaying twenty replicates of the zero standard. Comparison between the International Standard of AFP and standard serum of the new UMELISA format A linear regression analysis between the standard serum by independent concentrations and WHO International Standard 72/225 was performed. Correlation between the commercial assay and the new UMELISA format A correlation of 36 samples of low, medium and high concentrations was made, using the new UMELISA format and the Enzymun-Test AFP of Boehringer Mannheim (Code 1361236). Results and Discussion Standardization In this paper reagents of the new format and the former one, using UMELISA AFP are compared. Solid phase irradiated polystyrene plates (8 X 12 strips) and with titanium dioxide The stability of the reaction strips without their protective cover and stored with desiccant in the given bags was higher than 95% after 6 months. Table 1. Stability of the new reagents.
Points of Solid-phase (6 months) Tris 25x (2 months) the curve Control Strips %recovery Control R.T.U. %recovery --------------------------------------------------------------- 1 5.50 3.57 6.34 6.80 2 26.00 22.00 84.61 31.84 30.18 94.78 3 42.12 35.26 83.72 52.33 45.21 86.39 4 62.01 55.01 88.71 79.40 72.18 90.90 5 94.59 86.42 91.36 112.97 101.98 90.27 6 128.43 120.51 93.89 140.97 131.02 92.94 --------------------------------------------------------------- Points of Sheep serum (4 months) Conjugate (12 months) the curve Control R.T.U.% recovery Control R.T.U.% recovery --------------------------------------------------------------- 1 5.36 6.55 6.09 6.70 2 31.85 31.55 99.05 21.54 20.90 97.04 3 50.79 50.51 99.44 33.51 36.94 110.20 4 78.98 76.94 97.41 56.57 52.72 93.20 5 111.92 112.93 100.90 88.09 79.12 89.80 6 149.85 146.70 97.891 31.28 115.10 87.75 R.T.U. = Ready to use Tris Buffer 25X (liquid) It was diluted in distilled water and stored at 4 C during 2 months; stability was higher than 95%. Sheep serum 20X (liquid) It was diluted 1:4 with ready to use Tris buffer and was stored at 4 C during 4 months, then stability was above 95%. Table 2. Non-enzymatic hydrolysis of 4-MUP.
Time Relative fluorescence
(months) ----------------------
Concentrated Lyophilized
solution substrate
----------------------------------
0 0.90 1.21
18 1.80 32.15
Alkaline phosphatase-conjugate Anti-AFP (ready to use) The stability of the conjugate in the ready to use form was above 85% in 12 months. This presentation decreases laboratory mistakes. Table 1 presents the results obtained. Standard and control serum (lyophilized) Stability was higher than 90% after 12 months of reconstitution and storage at 4 C. Residual humidity in the lyophilized material was between 3 and 3.5%. The standard for independent concentrations prevents errors in the preparation of the calibration curve. Table 3. Standard curve of the new format (12 months).
Points of Control New format %recovery the curve --------------------------------------------- 1 5.33 5.59 2 23.23 21.90 94.27 3 34.26 32.10 93.69 4 56.60 54.42 96.14 5 87.34 81.97 93.85 6 123.54 118.14 95.62 Control 69.87 67.54 96.66 Statistical analysis Intercept = -0.69 Correlation = 0.9998 Slope = 1.0701 Angle = 44 degrees 4-MUP substrate (concentrated solution) The non-enzymatic hydrolysis of 4-MUP was checked by measuring relative fluorescence (Table 2). The recovery of the enzymatic activity in the assay was over 90%. Table 4. Precision.
Sample Intra-assay (n=30) Inter-assay (n=90)
X S.D. C.V. X S.D. C.V.
-----------------------------------------------------
Low 26.03 1.14 4.40 26.62 1.25 4.72
Medium 67.10 2.68 3.99 67.94 3.10 4.65
High 133.34 3.90 2.92 133.60 4.30 3.25
S.D. = standard deviation
C.V. = coefficient of variation
Standard curve Table 3 and Figure 1 show the standard curve obtained after 12 months of preparation. Precision The intra-assay (n=30) and inter-assay (n=90) reproduction was evaluated with 3 known specimens. Coefficients of variation obtained were below 5% (Table 4). The precision of the assay is adequate. Sensitivity Sensitivity of the new format is approximately 1.0 IU/mL. This was calculated as the concentration which was distinguishable from the zero standard, that is, two standard deviations above the fluorescence of the 0.00 IU/mL standard serum. Linear regression analysis In the comparison between the WHO International Standard 72/225 and the present standard, a correlation of 0.9996, a slope of 0.9798 and an angle of 44 degrees was obtained. Correlation assay The correlation coefficients for UMELISA versus Enzymun-Test AFP of Boehringer Mannheim was 0.9380. Screening in our country Table 5 shows a screening study of pregnant women started in Cuba in 1982 ; since then the data have remained the same. Table 6 shows the results for the introduction of this new UMELISA format in our country. Table 5. Screening in Cuba since 1982.
Number of pregnant women
-------------------------------------------------
Study of pregnant women 1 605 409
NTD diagnosis 2 024
Other malformations 1 288
Other diagnoses 11 007
Incidence X 1 000 2.06
Table 6. Screening with the introduction of the new format.
Number of pregnant women
-------------------------------------------------
Study of pregnant women 112 206
NTD diagnosis 91
Other malformations 47
Other diagnoses 284
Incidence X 1 000 1.20
Conclusions It can be concluded that with the introduction of these changes time was saved, special laboratory abilities are not required and there is a better use of the Kit. The new format has a higher precision and works better than the former one. Quantification is similar for new UMELISA AFP and Boehringer Mannheim format since the correlation coefficient obtained is 0.9380. 1. Ball D, Rose E and Alpert E. Alpha-fetoprotein levels in normal adults. The American Journal of the Medical Sciences 1992;303:157-159. 2. Manual de Diagnostico Prenatal para sanitarios Ed. Ministerio de Sanidad y Consumo, Madrid 1991. 3. Sundaram SG, Goldstein PJ, Manimekalai S and Wenk RE. Alpha- Fetoprotein and screening markers of congenital disease. Clin Lab Med 1992;12:481-492. 4. Milunsky A. Genetic disorders and the fetus: diagnosis, prevention and treatment, Ed. Plenum Press, N. York 1979 5. Morrow RJ, McNay MB and Whittle MJ. Ultrasound detection of Neural Tube Defects in patients with elevated Maternal Serum Alpha-Fetoprotein. Obstetrics and Gynecology 1991; 78:1055-1057. 6. Report of U.K. Collaborative. Study on Alpha-fetoprotein in relation to Neural Tube Defects: Maternal serum Alpha-fetoprotein measurement in antenatal diagnosis screening for anencephaly and spina bifida in early pregnancy. Lancet 1977;1:1323-1332. 7. Martinez-Frias ML. Defectos congenitos en Espana. Diez anos de vigilancia epidemiologica, De. Direccion general de planificacion sanitaria. Ministerio de Sanidad y Consumo, Madrid 1989. 8. Waller DK, Mills JL, Simpson JL, Gunningham GL, Conley MR, Lassman MR and Rhoads GG. Are obese women at higher risk for producing malformed off-spring. Am J Obstet Gynecol 1994;170:541- 548. 9. Greenberg F, del Junco D, Weyland B, Andrew W, Schmidt D, Rose E and Alpert E. The effect of gestational age on the detection rate of Down's syndrome by maternal serum Alpha- fetoprotein screening. Americam Journal of Obstetrics and Gynecology 1991; 165:1391-1393. 10. Aitken DA, McCaw G, Crossley JA, Berry E, Connor JM, Spencer K and Macri JN. First-trimester biochemical screening for fetal chromosome abnormalities and Neural Tube Defects. Prenat Diagn 1993;13:681-689. 11. Spencer K, Macri JN, Anderson RW, Aitken DA, Berry E, Crossley JA, Wood PJ, Coombes EJ, Stround M and Worthington DJ. Dual analyte immunoassay in Neural Tube Defect and Down's syndrome screening: results of a multicentre clinical trial. Ann Clin Biochem 1993;30:394-401. 12. Gadsden RH and Cate JC. Serum Alpha-fetoprotein: I. Evaluation of quantitative assays adapted to automated immunoassay systems. Annals of Clinical and Laboratory Science 1991;21:246-253. 13. Cate JC and Gadsden RH. Serum Alpha-fetoprotein: II. Clinical Evaluation of two automated immunoassays for maternal serum screening. Annals of Clinical and Laboratory Science 1991;21:254-257. 14. Masseyeff RF and Malvano R. Problems in standardization of Enzyme-immunoassays for antigens. [s.l:s.a:s.n] 38-53. 15. Solis RL, Fernandez JL, Robaina R, Heredero L and Rodriguez L. 10 years- Cuban Alpha-fetoprotein program. In: Libro de resumenes 8vo Simposio Internacional de Screening Neonatal y Encuentro Inaugural de la Sociedad Internacional de Screening Neonatal 1991;Resumen P78A. 16. Fernandez JL, Garcia FA, Solis RL, Robaina R, Garcia MA, Valdes V, Heredia L and Silva L The Ultramicroanalytic System (SUMA): A versatil technology for clinical testing. In: Libro de Resumenes Biotecnologia '91 1991:203. 17. Kautzmann M, Solis RL, Luberta A and Tanda R. UltramicroELISA for Detection and Quantification of Human Chorionic Gonadotrophin in Urine and Serum. Journal Clinical Ligand Assay 1995;18:121-125. 18. Fajardo EM, Fernandez JL, Solis RL, Portuondo B, Heredia L, Norona M, Urquiza H y Amat M. UltramicroELISA para medir antitoxina tetanica en suero humano. Boletin de la Oficina Sanitaria Panamericana 1995;119:113-120. 19. Kautzmann M, Solis RL, Luberta A, Fernandez JL, Navarro J, Rodriguez L, Hernandez J, Carrillo L, Llanuza C and Carrillo H. Study of the Efficiency of Screening for Trisomy 21 Based on Maternal Serum Level of AFPand HCG Combined with Maternal Age. Journal of Clinical Ligand Assay 1995;18:181-185. 20. Esser P and Rasmussen SE. Principles in adsorption to polystyrene. In: Nunc Bulletin 1988;6:1-8. 21. Avrameas S. Detection d' anticorps et d' antigenes a l'aide d' enzymes. Bull Soc Chim Biol 1968;50:1169. Copyright 1996 Elfos Scientiae
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