Measurement of Pregnancy-Associated Plasma Protein-A
Development of Production Technology of the Kit for Quantitative Immunoenzyme Measurement of Pregnancy-Associated Plasma Protein-A (РАРР-А) in Human Serum
Tech Area / Field
- MED-DID/Diagnostics & Devices/Medicine
3 Approved without Funding
State Research Center of Virology and Biotechnology VECTOR, Russia, Novosibirsk reg., Koltsovo
- Medical Institute of Postgraduate Training, Russia, Kemerovo reg., Novokuznetsk
- University of Leeds / Centre for Reproduction, Growth & Development, UK, Leeds
Project summaryAccording to World Health Organization (WHO) 2,5% of infants are born with congenital abnormalities. About 40-50% of early infant (perinatal) mortality and childhood disability is caused by congenital factors. Congenital abnormalities and chromosomal anomalies are most frequent diseases of children with mental deficiency. Perinatal mortality is one of the main indicators of healthcare level. Wide application of modern methods of fetus diagnostics in developed countries results in decreasing of perinatal mortality to 6-9‰. The same figure for Russia is 16-18‰, mainly because insufficient attention is given to application of modern methods of perinatal screening. Diagnostics of congenital anomalies in Russia is based on results of ultrasound scanning. Karyotypic analysis is conducted in special centers in very limited small quantities, which cannot solve the problem. Moreover, karyotypic analysis is invasive, therefore unsafe for a mother and a fetus, since fetus blood is required for the analysis. Imperfect screening for fetus congenital anomalies considerably increases quantity of ill children born. Thus frequency of infants with Down’s syndrome in Kemerovo region increased during 10 years from 0.42 in 1992 to 1.38 in 2002 for 1000 infants. In Novosibirsk region 16-17 children with Down’s syndrome are born annually.
According to the order of Russian Federation Ministry of Public Health N 457 on 20.12.00 screening of chromosomal disorders has been introduced into practical healthcare of Russian Federation, including determination of human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), and free (non-conjugated) estriol (NE) levels in serum of women at 15-20-th week of pregnancy. Efficiency of such “triple test” is 60-70%, but it is diagnostically important only since the second pregnancy trimester (15-20-th week), when abortion operation may lead to various severe complications, to the extent of sterility. Therefore there is understandable aspiration of researchers to discover diagnostic criteria for fetus anomalies detection in early pregnancy period, when induced abortion is not so hazardous.
Pregnancy-associated plasma protein-A (PAPP-A) was detected in pregnant women serum in 1974 for the first time. This is a high-molecular metalloprotein (m.m. 820 kDa) with 2α-electrophoretic mobility, which belongs to the macroglobulin family. Significant decrease of pregnancy-associated plasma protein-A (PAPP-A) level in the first pregnancy trimester associated with Down’s syndrome (DS) was firstly detected in 1991. Furthermore it was shown that determination of PAPP-A and β-subunit of human chorionic gonadotropin (β-HCG) levels on 10-11 week of pregnancy, considering mother’s age, and scanning of collar area (by ultrasound) allows detection of Down’s syndrome with 89% efficiency. Clinicians have been showing more and more interest in PAPP-A for the last years because of its high self-descriptiveness for early detection of Down’s syndrome – on 10-11 week of pregnancy. It was shown that decreased PAPP-A level can be associated not only with Down’s syndrome (trisomy 21) but also with other severe chromosomal anomalies of fetus. That is Edward’s syndrome (trisomy 18), Patau syndrome (trisomy 13), Turner’s syndrome (monosomy X), triploidy and aneuploidy of sex chromosomes. Detectability of these diseases by measuring PAPP-A and β-HCG levels on 10-13 week of pregnancy and considering US data amounts to 87%. It was also demonstrated that decreased PAPP-A level in the first trimester is prognostic concerning following unfavorable pregnancy outcomes: ectopic pregnancy, spontaneous abortion, fetus growth inhibition, placenta rupture, stillbirth, and others. From the other hand, normal PAPP-A level in serum of women, being observed under threat of abortion in the first trimester, was associated with favorable pregnancy outcome in 99% of cases.
Percentage of infertile marriages in Russia amounts to 17% and has a tendency to increase. Introducing of extracorporal fertilization (ECF) made a breakthrough in infertility treatment, which made possible implementing reproductive function under such infertility forms that had been considered hopeless for treatment. It was shown that measurement of PAPP-A concentration in serum is essentially important for observation of postimplantation development of artificially fertilized ovum. Women who had low PAPP-A serum content in the first trimester of ECF-induced pregnancy would later have reliably higher probability of preterm delivery. It was also shown, that PAPP-A is more reliable marker of ECF-induced pregnancy, since increase of TBG (trophoblastic β-1-glycoprotein) and HCG levels does not exclude later fetal ovum rejection. Moreover, increased HCG level may be caused by exogenous protein present in blood. Therefore, increase of PAPP-A level after artificial fertilization may be used as a prognostic index for operation efficiency.
Hence it is proved that pregnancy-associated plasma protein-A (PAPP-A) is a highly informative protein marker of chromosomal fetal abnormalities and other pregnancy disorders. Nowadays PAPP-A level measurement is obligatory in many countries (Germany, Great Britain, Switzerland and others) during screening for chromosomal fetal abnormalities in the early pregnancy period. At present there is no kit for PAPP-A measurement in Russia, permitted for medical application. Considering increase of congenital abnormalities of Russian infants and lack for high-quality diagnostics, we propose to develop and subsequently introduce into medical practice a kit for quantitative PAPP-A measurement. The future developed kit is planned to be effective, comparing with best foreign analogues, but much less expensive which is very important for Russian consumers. Introduction of this kit into standard diagnostic procedures will significantly increase the efficiency of prenatal screening for fetal abnormalities.
A method to produce small amount of high-purity PAPP-A, sufficient for research, has been developed in Medical Institute of Postgraduate Training (MIPT) in Novokuznetsk. Isolated pregnancy-associated plasma protein-A (PAPP-A) retains its native form and is free from admixture of other serum proteins and homological macroglobulins. Immunization of animals with PAPP-A provides us with polyclonal antiserum to this protein, containing highly specific antibodies to all the epitopes of natural PAPP-A molecule. Development of technology for producing PAPP-A and highly specific polyclonal antibodies to PAPP-A in sufficient quantities for serial production of the kit for quantitative PAPP-A measurement is planned within the framework of the project.
Highly sensitive and specific immunometric method for measurement of human chorionic gonadotropin (HCG) has been developed in SRC VB “Vector” using two types of polyclonal antibodies specific to different HCG antigenic determinants [patent N 2062473, Russia]. The method is based on monospecific antibodies to HCG, obtained with the help of synthesized peptide corresponding to unique C-end aminoacid sequence of HCG β-subunit 115-145, and antibodies to antigenic determinants located outside this region. Based on this method the kit “PREGNATEST-ELISA-HCG-1” for immunoenzyme measurement of human chorionic gonadotropin was developed and approved for medical application by Committee for new medical equipment of Russian Federation Ministry of Public Health (TU 9398-332-23548172-97). Serial production of the kit has been established, and it is widely used in practical healthcare in Russia and CIS states.
It is known that aminoacid sequence of pregnancy-associated plasma protein-A (PAPP-A) is unique over the significant length. Within proposed project it is planned to develop method for producing monospecific antibodies to PAPP-A using synthesized peptides corresponding unique parts of PAPP-A aminoacid sequence.
We propose a project to develop technology of kit production for quantitative immunoenzyme measurement of PAPP-A using highly specific polyclonal antibodies, produced in Novokuznetsk MIPT, in combination with monospecific antipeptide antibodies, production method of which will be developed in SRC VB “Vector”.
The project goals are the following:
- To obtain and characterize highly purified pregnancy-associated plasma protein-A (PAPP-A), free from admixtures of other serum proteins and homological macroglobulins.
- To isolate highly specific polyclonal antibodies to pregnancy-associated plasma protein-A (PAPP-A).
- To synthesize and characterize peptides corresponding to the unique regions of PAPP-A aminoacid sequence.
- To isolate monospecific antibodies to synthesized peptides.
- To produce the components and construct the kit for quantitative immunoenzyme measurement of pregnancy-associated plasma protein-A (PAPP-A) in human serum.
- To manufacture experimental series of the developed kit and compare it with commercial analogues. To approve the developed kit in laboratory conditions using serum specimens from pregnant women and oncological patients.
- To prepare the complete set of scientific and technological documentation (STD) for the kit for quantitative immunoenzyme measurement of pregnancy-associated plasma protein-A (PAPP-A) in human serum.
- To conduct medical trials of the developed kit in medical institutions of Siberian region and Moscow.
Widespread introduction of the proposed kit into healthcare practice will allow establishing mandatory measurement of PAPP-A serum levels for pregnant women in Russia in the first trimester to early detect such congenital fetus abnormalities as Down’s syndrome, Edward’s syndrome, and others. Monitoring of PAPP-A during the whole pregnancy will provide detection of threatened-miscarriage pregnancy, spontaneous abortion, fetus growth inhibition, ectopic pregnancy and prediction of pregnancy outcome. Measurement of PAPP-A maternal serum level during the extracorporal fertilization-induced pregnancy will make possible implementing reproductive function for women with such infertility forms that were so far considered absolutely hopeless for treatment. Implementation of PAPP-A measurement may be useful in oncology as diagnostic and prognostic criterion for ovarian cancer and in cardiology as an additional biomarker of coronary artery disease.
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