Interfacial structure of immobilized antibodies and perdeuterated HSA in model pregnancy tests measured with neutron reflectivity.

Experimental studies of antibody adsorption and antigen binding that mimicked pregnancy test immunoassays have been performed using neutron reflectivity studies of a model antibody/antigen system immobilized on the silica/water interface. The study revealed the nature of the antibody/antigen interaction and also the importance of a blocking protein, in this case human serum albumin (HSA), that enhances the immunoassay's specificity and efficiency. Of central importance to this study has been the use of a perdeuterated human serum albumin (d-HSA), providing contrast that highlights the orientation and position of the blocking agent within the adsorbed layer. It was found that the adsorbed HSA filled the gaps between the preadsorbed antibodies on the substrate, with decreased adsorption occurring as a function of increased antibody surface coverage. In addition, the antigen binding capacity of the adsorbed antibodies was investigated as a function of antibody surface coverage. The amount of specifically bound antigen was found to saturate at approximately 0.17 mg/m(2) and became independent of the antibody surface coverage. The ratio of bound antigen to immobilized antibody decreased with increased antibody surface coverage. These results are of importance for a full understanding of immunoassay systems that are widely used in clinical tests and in the detection of environmental contaminants.

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Substituting whole blood for urine in a bedside pregnancy test.

BACKGROUND: Point-of-care testing for rapid detection of pregnancy in women of reproductive age is common practice in the emergency department. Commercially available rapid human chorionic gonadotropin (hCG) immunoassays are validated for use with urine and serum, but not whole blood. STUDY OBJECTIVES: We assessed the validity of using whole blood to detect pregnancy using a point-of-care hCG assay by comparing it to a laboratory quantitative serum hCG assay as the criterion standard. METHODS: A convenience sample of female patients of reproductive age (18-51 years) submitted 5mL of whole blood, from which two drops were immediately applied to a point-of-care hCG kit, with results recorded at 10min. The remainder of each whole blood specimen was sent to the hospital laboratory for the criterion-standard quantitative serum hCG assay. The criterion standard for a positive pregnancy test was defined as quantitative serum hCG≥5 mIU/mL. Investigators performing the whole blood test and laboratory technicians performing the quantitative serum assay were blinded to one another's results. RESULTS: There were 633 patients enrolled, with a mean age of 30 years (± 7.7 years); 34% of the patients were pregnant. Overall, the whole blood pregnancy test was 95.8% sensitive (negative predictive value 97.9%), whereas the urine test was 95.3% sensitive (negative predictive value 97.6%); the specificity and positive predictive value of both tests was 100%. CONCLUSION: Using a standard point-of-care qualitative hCG immunoassay kit, whole blood may be used for rapid detection of pregnancy with similar, or greater, accuracy than urine.

Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows.

The objective of the present study was to compare two commercially available blood-based pregnancy tests, namely BioPRYN, an ELISA for pregnancy-specific protein B (PSPB), and an ELISA for pregnancy-associated glycoprotein (PAG), for early pregnancy diagnosis in dairy cattle using transrectal ultrasonography as a gold standard. Transrectal ultrasonography was conducted 26-58 days after artificial insemination (AI) in 197 cattle from 19 farms. Concurrently, a blood sample was collected for determination of serum PSPB and PAG. Transrectal palpation was performed approximately 120 days after AI to verify that pregnancy was maintained. For PSPB and PAG, there were no significant differences (P>0.05) in sensitivity (98.0 and 97.8%), specificity (97.1 and 91.2%), positive predictive values (99.3 and 97.8%), negative predictive values (91.9 and 91.2%) and accuracy (97.8 and 96.4%). In conclusion, the two blood pregnancy assays were equally efficacious and were highly accurate (based on transrectal ultrasonography as the gold standard).

Immunological detection of pregnancy: Evidence for systemic immune modulation during early pregnancy in ruminants.

Mammalian pregnancy creates unique challenges for immune systems highly evolved to detect and eliminate invading pathogens. Recognition of the challenges created by gestating a semi-allogeneic fetus evolved from the discipline of transplantation biology and were informed by studies on the unique natural parabiosis that occurs when female calves are gestated with twin male fetuses. These pregnancies typically result in an intersex female termed a freemartin, which revealed insights into development of the male and female reproductive tracts. However, they also uncovered important clues on immune tolerance with wide-ranging implications to reproductive biology, transplantation biology and autoimmune disease. Many studies focused on identifying mechanisms through which the fetus evades maternal immune detection and elimination. These included studies characterizing immune interactions between the fetus and mother at the nourishing interface of the placenta and uterine endometrium. This immunological forbearance only occurs under high concentrations of circulating progesterone. Beyond the requirement for progesterone, there has been considerable progress towards understanding the effects of conceptus signals on maternal immune function. One common theme is that pregnancy induces a T helper 2 immune bias as shown in several mammalian species, including domestic ruminants. However, a growing body of evidence shows that the fetus not only evades, but also provokes immune responses locally in the uterus and in peripheral tissues. This is perhaps most dramatically illustrated by domestic ruminants where the conceptus secretes a unique interferon in the opening salvo of hormonal communication with the maternal immune system. The role of interferon tau in regulating expression of genes of the innate immune system in the uterus has been extensively studied. More recently, it was determined that these same genes are also induced in peripheral immune cells and other tissues throughout the body. In addition to interferon tau and progesterone, pregnancy associate glycoproteins and chaperonin 10 (aka Early Pregnancy Factor) are implicated in altering immune function both locally and systemically during pregnancy. While it is tempting to speculate that this activation of innate immunity is designed to counteract selective immunosuppression, knowledge of the importance of local and systemic immune activation to the success of pregnancy remains incomplete. This area remains fertile ground for developing better approaches to diagnose and treat infertility in domestic farm species and humans alike.

Screening for twin pregnancy.

A group of 590 women who, 4 to 5 weeks after their last menstrual period, were confirmed to be pregnant, as measured by the human chorionic gonadotropin (hCG) by radioreceptor assay. Nine of these women had serum hCG levels approximately twofold higher than the others and were suspected of having twin pregnancy. When these women were tested at 12 weeks of gestation, pelvic sonography confirmed twin pregnancies in all the nine cases. Serum hCG levels thus provide a simple, rapid, and easy method to detect twin pregnancy.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle.

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Measurement of EPF for detection of cow pregnancy using rosette inhibition test.

Early embryonic death of calves due to sub-fertility in cows is of great economic concern to dairy industry. Early pregnancy factor (EPF) is a secretory protein with pregnancy associated immunosuppressive properties. Rosette inhibition test (RIT) was used to detect EPF in inseminated dairy cows. Blood samples were collected at two intervals, 1-3 and 5-7 days after insemination from 23 inseminated and 18 non-inseminated control cows for RIT and pregnancy diagnosis performed between 42 and 45 days on palpation. The study indicates that RIT (P<0.05) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy.

Field experiences with early pregnancy diagnosis by progesterone-based ELISA in sows.

In four Kenyan pig breeding units the pregnancy diagnosis of sows has been carried out in two groups: Group 1 (n = 1911): the sows were transrectaly pregnancy tested between Days 17-22 post-mating by ultrasound. Sows testing non-pregnant immediately received one dose of 400 IU pregnant mare serum gonadotropin (PMSG) (equine chorion gonadotropin, eCG) and 200 IU human chorion gonadotropin (hCG). On showing signs of oestrous, the animals were subsequently artificially inseminated (AI). Group 2 (n = 1923): sows were pregnancy tested by serum progesterone (P4)-based enzyme-linked immunosorbent assay (ELISA) on Day 17 post-breeding. P4 concentrations were categorized as positive (> 5 ng/ml) or negative (< 5 ng/ml). Sows testing nonpregnant immediately received one dose of 400 IU PMSG and 200 IU hCG by injection, and were subsequently artificially inseminated. The following parameters were evaluated: sows diagnosed non-pregnant, days from first post-weaning insemination until the sows were inseminated at their first return to oestrus; farrowing rate and total piglets born and number of live-born piglets in litters. The percentage of sows diagnosed non-pregnant in the two groups, as well as the totals of born piglets and of live-born piglets in litters did not differ significantly between the two groups. The number of days from the first post-weaning mating until the sows were artificially inseminated at their first return to oestrus and the administration of eCG and hCG was shorter (P < 0.01) and farrowing rate was higher (P< 0.01) in the ELISA-tested sows.

Quantification of urinary chorionic gonadotropin in spontaneous abortion of pre-clinically recognized pregnancy: method development and analytical validation.

Determination of environmental impacts on reproductive health and specifically on the incidence of early spontaneous abortion requires accurate estimates of the latter. This negative reproductive outcome can be detected by the pattern of elevation and decline of human chorionic gonadotropin (hCG) levels near and shortly beyond the expected time of implantation, requiring daily biomonitoring of hCG levels during the relevant period of the menstrual cycle. Prospective pregnancy studies to assess effects of potentially toxic exposures on human reproductive outcomes can involve up to three menstrual cycles and a huge number of samples in each, for the quantification of the inherently very low hCG levels usually can be determined only in serum. The invasive nature of blood collection, the number of samples needed for the development of prospective studies, and the lack of quantitative methods for the determination of low hCG levels in urine point to the need for collecting urine rather than blood and make it imperative to develop suitable quantitative methods for biomonitoring of very low levels of hCG in urine. This paper describes the development and validation procedures of an automated solid-phase two-site chemiluminescent immunometric assay for the quantification of urinary hCG in early pregnancy and early pregnancy loss. For the validation, both undiluted and diluted urine and control samples have been prepared. From the results, it can be concluded that the assay has a calibration range that extends to 5000 mIU/ml, with a detection limit of approximately 1.2 mIU/ml, practically identical to that found by the IMMULITE 2000 manufacturer's validation study. The intra- and inter-assay precision ranges up to a maximum of around 7%, meaning that the practical limit for functional sensitivity can be established as low as 10%. This means that the immunoassay from DPC can identify, with relatively high confidence, non-pregnant women and the typical "rise and fall" pattern of early pregnancy loss through analysis of urine samples. Results also lead to the conclusion that there is a very good agreement between expected and observed urinary hCG levels indicative of good immunoassay accuracy for the studied range of hCG concentrations. In terms of analyte stability, it can be concluded that urinary hCG is stable under the expected conditions required for ongoing investigations that include temperatures of 2-8 degrees C for up to 48 h and temperatures of around -20 degrees C for longer periods that can extend to over 3 months.

Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial insemination.

To determine the accuracy of a pregnancy-associated glycoprotein (PAG) ELISA in identifying pregnancy status 27 d after timed artificial insemination (TAI), blood samples were collected from lactating Holstein cows (n = 1,079) 27 d after their first, second, and third postpartum TAI services. Pregnancy diagnosis by transrectal ultrasonography (TU) was performed immediately after blood sample collection, and pregnancy outcomes by TU served as a standard to test the accuracy of the PAG ELISA. Pregnancy outcomes based on the PAG ELISA and TU that agreed were considered correct, whereas the pregnancy status of cows in which pregnancy outcomes between PAG and TU disagreed were reassessed by TU 5 d later. The accuracy of pregnancy diagnosis was less than expected when using TU 27 d after TAI (93.7 to 97.8%), especially when pregnancy outcomes were based on visualization of chorioallantoic fluid and a corpus luteum but when an embryo was not visualized. The accuracy of PAG ELISA outcomes 27 d after TAI was 93.7, 95.4, and 96.2% for first, second, and third postpartum TAI services, respectively. Statistical agreement (kappa) between TU and the PAG ELISA 27 d after TAI was 0.87 to 0.90. Pregnancy outcomes based on the PAG ELISA had a high negative predictive value, indicating that the probability of incorrectly administering PGF(2alpha) to pregnant cows would be low if this test were implemented on a commercial dairy.

Evaluation of early conception factor lateral flow test to determine nonpregnancy in dairy cattle.

The early conception factor (ECF) lateral flow test was evaluated for its ability to accurately determine nonpregnant status in dairy cattle. Results of 2 field trials involving 191 cows and 832 tests indicated the probability that a cow can be correctly diagnosed as nonpregnant by using the ECF test is only about 50%. Agreement of test results between milk and serum obtained from the same cow was 57.5%. The ECF test was not consistent in identifying nonpregnancy when the same cows were tested repeatedly over a period of 4 weeks. We conclude that the ECF lateral flow test does not accurately identify nonpregnancy in dairy cattle.

Reducing False-Positive Pregnancy Test Results in Patients With Cancer.

OBJECTIVE: To assess whether the use of a laboratory test specific for intact human chorionic gonadotropin (hCG) would reduce the number of false-positive pregnancy test results. METHODS: From October 21, 2014, to January 20, 2015, and April 1, 2015, to June 2, 2015, all serum samples sent for pregnancy screening at a large cancer center with a value of 5 milli-international units/mL or greater total ß-hCG were frozen and stored and then retested using intact hCG reagent. We compared the accuracy of total ß-hCG and intact hCG results for the diagnosis of clinically confirmed pregnancy. A negative test was defined as 14 milli-international units/mL or less, our current institutional cutoff. We also assessed a cutoff of less than 5 milli-international units/mL, a historical cutoff to rule out pregnancy. RESULTS: We performed intact hCG testing on 64 patient samples, of which 34 had originally resulted positive when tested for total ß-hCG. These included 21 cases of clinically confirmed pregnancy and 13 false-positive cases. No women were pregnant when their intact hCG concentration was 14 milli-international units/mL or less, and all pregnancies were detected at and above this concentration. Intact hCG reduced the number of false-positive pregnancy test results from 13 to 1, a 92% reduction (95% CI 64-99%), corresponding to a reduction in the false-positive rate from 38% (95% CI 22-56%) to 3% (95% CI 1-15%). CONCLUSION: The use of intact hCG reagent in patients with cancer reduces the rate of false-positive pregnancy test results without increasing the rate of false-negative test results.

A single serum test for measuring early pregnancy outcome with high predictive value.

OBJECTIVES: Current testing to determine a failing pregnancy requires two separate clinic visits to measure the hCG doubling rate. Diagnosing a failing pregnancy is often done in emergency departments where simplified and accelerated testing methods are needed. Here, we investigated hyperglycosylated hCG (hCG-H) for predicting pregnancy failure. DESIGN AND METHODS: We studied two independent sets of patient samples collected in the early weeks of gestation. One set was urine samples, and the other was serum samples. In all cases, hCG and hCG-H were measured using automated chemiluminescence immunoassays. Concentrations of hCG and hCG-H were plotted on a scattergram, and levels in failing pregnancies were compared to those in continuing pregnancies. RESULTS: Data indicated that a threshold level of hCG-H (13 microg/L) in both serum and urine samples defined the concentration below where pregnancies were likely to fail. This cut-off corresponded to 73% detection of failures at a 2.9% false positive rate using serum and 75% detection at a 15% false positive rate using urine. Using an hCG cut-off that corresponded to the same false positive rates, hCG detected only 42% of failures using serum and 43% of failures using urine. CONCLUSIONS: Our data indicate that hCG-H provides a much more accurate single test than hCG for assessing pregnancy outcome. Compatible with the use of serum or urine samples, a single hCG-H test might provide simpler, faster, and more accurate results for predicting the progress of a pregnancy than standard hCG testing.

Comparison of Result Times Between Urine and Whole Blood Point-of-care Pregnancy Testing.

INTRODUCTION: Point-of-care (POC) pregnancy testing is commonly performed in the emergency department (ED). One prior study demonstrated equivalent accuracy between urine and whole blood for one common brand of POC pregnancy testing. Our study sought to determine the difference in result times when comparing whole blood versus urine for the same brand of POC pregnancy testing. METHODS: We conducted a prospective, observational study at an urban, academic, tertiary care hospital comparing the turnaround time between order and result for urine and whole blood pregnancy tests collected according to standard protocol without intervention from the investigators. After the blood was collected, the nurse would place three drops onto a Beckman Coulter ICON 25 Rapid HCG bedside pregnancy test and set a timer for 10 minutes. At the end of the 10 minutes, the result and time were recorded on an encoded data sheet and not used clinically. The same make and model analyzer was also used for urine tests in the lab located within the ED. The primary outcome was the difference in mean turnaround time between whole blood in the ED and urine testing in the adjacent lab results. Concordance between samples was assessed as a secondary outcome. RESULTS: 265 total patients were included in the study. The use of whole blood resulted in a mean time savings of 21 minutes (95% CI 16-25 minutes) when compared with urine (p<0.001). There was 99.6% concordance between results, with one false negative urine specimen with a quantitative HCG level of 81 mIU/L. CONCLUSION: Our results suggest that the use of whole blood in place of urine for bedside pregnancy testing may reduce the total result turnaround time without significant changes in accuracy in this single-center study.

Quality assurance for multiplexed assays - how can it be achieved?

Multiplexed assays are now a common form of analysis in routine clinical and research laboratories. Assuring the quality of this type of complex, massively-parallel testing poses challenges not encountered in the traditional single-plex assay. A range of quality assurance measures is implemented at different stages in a multiplex assay, beginning in the manufacturing process, and the ensuing analytical and data interpretation stages. This article explores quality issues and the quality assurance measures that have been devised for multiplex assays ranging from simple two-plex assays to the types of assays that involve simultaneous testing on millions of test sites on a single analytical device.

Quantitative analysis of total ß-subunit of human chorionic gonadotropin concentration in urine by immunomagnetic reduction to assist in the diagnosis of ectopic pregnancy.

BACKGROUND: The initial diagnosis of ectopic pregnancy depends on physical examination, ultrasound, and serial measurements of total ß-subunit of human chorionic gonadotropin (hCGß) concentrations in serum. The aim of this study was to explore the possibility of using quantitative analysis of total hCGß in urine rather than in serum by immunomagnetic reduction (IMR) assay as an alternative method to diagnose an ectopic pregnancy. METHODS: We established a standard calibration curve of IMR intensity against total hCGß concentration based on standard hCGß samples, and used an IMR assay to detect total hCGß concentrations in the urine of pregnant women with lower abdominal pain and/or vaginal bleeding. The final diagnosis of ectopic pregnancy was based on ultrasound scans, operative findings, and pathology reports. In this prospective study, ten clinical samples were used to analyze the relationship of total hCGß IMR signals between urine and serum. Furthermore, 20 clinical samples were used to analyze the relationship between urine IMR signals and serum levels of total hCGß. RESULTS: The calibration curve extended from 0.01 ng/mL to 10,000 ng/mL with an excellent correlation (R(2)=0.999). In addition, an excellent correlation of total hCGß IMR signals between urine and serum was noted (R(2)=0.994). Furthermore, a high correlation between urine IMR signals and serum levels of total hCGß was noted (R(2)=0.862). CONCLUSION: An IMR assay can quantitatively analyze total hCGß concentrations in urine, and is a potential candidate for point-of-care testing to assist in the diagnosis of ectopic pregnancy.

Direct Reading of Bona Fide Barcode Assays for Diagnostics with Smartphone Apps.

The desire to develop new point-of-care (POC) diagnostic tools has led to the adaptation of smartphones to tackle limitations in state-of-the-art instrumentation and centralized laboratory facilities. Today's smartphones possess the computer-like ability to image and process data using mobile apps; barcode scanners are one such type of apps. We demonstrate herein that a diagnostic assay can be performed by patterning immunoassay strips in a bona fide barcode format such that after target binding and signal enhancement, the linear barcode can be read directly with a standard smartphone app. Quantitative analysis can then be performed based on the grayscale intensities with a customized mobile app. This novel diagnostic concept has been validated for a real-world application, i.e., the detection of human chorionic gonadotropin, a pregnancy hormone. With the possibility of multiplex detection, the barcode assay protocol promises to boost POC diagnosis research by the direct adaptation of mobile devices and apps.