Monoclonal Antibody-based Assays Research Articles

Clinical validation of renin monoclonal antibody-based sandwich assays of renin and prorenin, and use of renin inhibitor to enhance prorenin immunoreactivity

Newly developed IRMAs to measure the plasma concentrations of renin and prorenin were validated for clinical use and compared with a classical enzyme kinetic assay. The IRMAs involve two monoclonal antibodies, one that reacts equally well with renin and prorenin and one that recognizes renin well but prorenin only minimally. Prorenin reactivity with the second antibody was enhanced by adding the renin inhibitor, Remikiren, to plasma. The complex of prorenin with this active-site ligand undergoes a conformational change, whereby prorenin is converted into a form that cannot be differentiated from renin by the IRMA. The linear working range of the assay was 4.0-3000 mU/L. The concentration of prorenin was calculated by subtracting the assay result obtained without Remikiren (i.e., renin) from the result obtained with Remikiren (i.e., renin plus prorenin). No more than 2% of prorenin present in plasma was detected as renin. The interassay CVs for renin quantification were 18%, 13%, and 8% at low, medium, and high concentrations, respectively. The interassay CV for calculated prorenin was 8% at both low and high concentrations. The IRMA results were highly correlated with those of an enzyme kinetic assay in healthy subjects; in patients with such conditions as primary hyperaldosteronism, renovascular hypertension, and low-, medium-, and high-renin essential hypertension; and in women undergoing gonadotropin stimulation.

Plasminogen Activator Inhibitor (PAI-1) and Non-insulin-dependent Diabetes in Pima Indians, South Asians and Europeans

Activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of fibrinolysis, is raised in non-insulin-dependent diabetes mellitus (NIDDM) and may contribute to excess macrovascular disease. We investigated the association of PAI-1 activity with NIDDM in Pima Indians, a racial group with low risk of coronary artery disease, but in whom the impact of NIDDM on the occurrence of coronary artery disease is similar to other racial groups. We studied 308 (136 non-diabetic, 172 diabetic) Pima Indians from Arizona, and 138 (98 non-diabetic, 40 diabetic) South Asians and 129 (80 non-diabetic, 49 diabetic) Europeans living in North London. PAI-1 activity was measured by a spectrophotometric assay, and insulin, intact proinsulin and des 31,32 proinsulin concentrations were measured employing highly specific monoclonal antibody-based assays. Compared with non-diabetic subjects, PAI-1 activity was significantly higher in subjects with NIDDM among South Asians (22.8 +/- 7.3 vs. 17.4 +/- 6.9 AU/ml, p < 0.001) and Europeans (23.1 +/- 6.6 vs. 16.5 +/- 6.1 AU/ml, p < 0.001) but not among Pima Indians (19.5 +/- 9.6 vs. 18.5 +/- 8.6 AU/ml, ns). The association of PAI-1 activity with diabetes remained statistically significant when controlled for age, sex, body mass index and waist-hip ratio, serum triglyceride and fasting insulin concentrations in South Asians (p < 0.001) and Europeans (p < 0.001). The relationships of PAI-1 activity with clinical and biochemical variables were similar in the three racial groups other than for fasting and 2 h plasma glucose concentrations which were significantly associated with PAI-1 activity in South Asians (partial r = 0.32 and 0.31) and Europeans (partial r = 0.44 and 0.50) but not in Pima Indians (partial r = 0.11 and 0.15). In Pima Indians with NIDDM, PAI-1 activity was similar in those treated with sulphonylureas, insulin, or no drugs for diabetes. In conclusion, the association of PAI-1 with diabetes differs in racial groups who are at different risk of NIDDM and ischaemic heart disease. Previously reported differences in the prevalence of ischaemic heart disease between diabetic and non-diabetic Pima Indians, and between non-diabetic subjects from the three racial groups, are unlikely to be due to differences in PAI-1 activity.

Cerebrospinal fluid levels of β endorphin in painful and painless diabetic polyneuropathy

β endorphin (β-EP) is an important modulator of central pain pathways. To examine whether changes in central production of β-EP contribute to the pathogenesis of diabetic neuropathic pain, we compared the cerebrospinal fluid (CSF) levels of β-EP and its precursor proopiomelanocortin (POMC) between 15 diabetic patients with chronic painful diabetic polyneuropathy, eight patients with severe painless diabetic neuropathy, and ten nondiabetic controls. Both peptides were measured by specific monoclonal antibody-based two-site immunoradiometric assays (IRMAs). In the diabetic patients with painful neuropathy, mean ± SD CSF β-EP concentrations (5.7 ± 2.2 pmol/L) were comparable to those of the diabetic patients with painless neuropathy (6.0 ± 2.3 pmol/L) and did not correlate with the severity of neuropathic pain. CSF β-EP, but not POMC, concentrations were lower in the diabetic neuropathic patients overall (5.8 ± 1.9 pmol/L) compared to the control subjects (7.6 ± 2.2 pmol/L) ( p < 0.05). CSF POMC showed no intergroup differences. However, POMC levels were 80-fold higher than those of β-EP and should always be considered when interpreting immunoreactive β-EP or other derivative peptide levels in CSF. We conclude that CSF β-EP levels appear to be reduced in diabetic polyneuropathy but they do not relate to the presence of neuropathic pain. This might explain why opioid analgesics are of little, if any, help in alleviating diabetic neuropathic pain.

IgA, IgG and IgM quantification in bronchoalveolar lavage fluids from allergic rhinitics, allergic asthmatics, and normal subjects by monoclonal antibody-based immunoenzymetric assays

Recent reports have suggested that human secretory IgA (sIgA) may have a role in the mediation of atopic disease. We have studied the levels of sIgA, IgA, IgG and IgM in bronchoalveolar lavage (BAL) fluids collected from lungs of healthy non-allergic adults ( n = 14), allergic subjects with rhinitis ( n = 15), and allergic asthmatics ( n = 13), using a panel of monoclonal antibody-based immunoenzymetric assays (IEMAs). In contrast to commercially available immunodiffusion and nephelometric assays, these IEMAs employ highly specific monoclonal antibodies and demonstrate required precision (intra-assay CVs < 17%), parallelism (inter-dilutional CVs < 20%) at minimal detectable immunoglobulin levels in the ng/ml range, and excellent specificity with < 0.1% crossreactivity for heterologous immunoglobulin isotypes. Using these assays, we have observed a significant correlation between sIgA levels and total IgA levels in BAL fluids from all the study patients ( r = 0.94; p < 0.01). The percentage of sIgA to total IgA was 84.0 ± 2.2%. sIgA in BAL fluids from allergic rhinitics (18.0 ± 2.5 μg/ml) and allergic asthmatics (15.5 ± 2.5 μg/ml) were higher than those from nonallergic subjects (10.2 ± 1.9 μg/ml). The only statistically significant difference in sIgA levels was observed in BAL fluids from the rhinitics and nonallergic groups ( p = 0.03). Similar differences among the groups were found for levels of total IgA in BAL fluid. There were no significant differences in the levels of IgM and IgG in BAL fluids among the three groups of subjects. We conclude from these results that IgA is the predominant immunoglobulin on the airway surface and that it appears to be produced locally.

Detection of feline immunodeficiency virus p24 antigen and p24-specific antibodies by monoclonal antibody-based assays

A panel of monoclonal antibodies (mAbs) detecting distinct B-cell epitopes on p24 core viral protein of feline immunodeficiency virus (FIV) were employed to develop immunoassays to measure p24 concentration in culture and serum samples, to localize p24 in FIV-infected cells and tissues, and to detect anti-p24 antibodies in cat sera. In its optimized configuration the p24 capture assay detected as little as 0.25 ng/ml of protein. The assay was found at least as sensitive as the reverse transcriptase activity assay in FIV-infected lymphocyte cultures and proved capable of detecting p24 antigen in acid pretreated sera from a high proportion of FIV-infected cats. The mAbs were also successfully used to detect the p24 antigen in permeated FIV-infected cells by flow cytometry and in tissue sections from FIV-infected cats by immunohistochemical staining. Anti-p24 antibodies in FIV-infected cat sera were assayed by a competitive capture ELISA which readily identified occasional false positive results provided by a standard ELISA using purified whole FIV-coated wells.

Effects of octreotide on circulating islet B cell products in endogenous hyperinsulinism

The role of somatostatin analogues in the medical management of insulinomas is unclear. We describe an elderly patient with clinical and biochemical features of endogenous hyperinsulinism attributable to a benign islet B cell disorder whose incapacitating neuroglycopaenic symptoms responded dramatically to octreotide 50 micrograms subcutaneously at 2200 h each night. Octreotide suppressed inappropriate plasma concentrations of insulin thereby preventing fasting hypoglycaemia. Fasting concentrations of proinsulin, and 32-33 split proinsulin, as determined by two-site monoclonal antibody-based immunoradiometric assays, were also suppressed by octreotide.

Hyperproinsulinaemia in impaired glucose tolerance.

1. Basal circulating concentrations of islet B cell products were measured using two-site monoclonal antibody-based immunoradiometric assays after a 10 h overnight fast in a group of non-obese subjects with recently diagnosed impaired glucose tolerance (World Health Organization criteria). A group of healthy subjects with normal oral glucose tolerance matched for age and body mass index served as normal controls. 2. Fasting blood glucose concentration was normal in all subjects with mean (+/- SEM) levels of 5.1 +/- 0.2 and 4.8 +/- 0.2 mmol/l (P > 0.1) for the group with impaired glucose tolerance and the healthy control group, respectively. 3. There was no significant difference (P > 0.1) in fasting plasma insulin or C-peptide concentrations between the groups. 4. By contrast, the fasting concentration of intact proinsulin was nearly four-fold higher in the subjects with impaired glucose tolerance than in the matched healthy control subjects (4.5 +/- 1.0 versus 1.2 +/- 0.2 pmol/l, P < 0.005). 5. Similarly, the fasting plasma concentration of 32-33 split proinsulin in the subjects with impaired glucose tolerance was almost twice that of the control subjects (7.4 +/- 1.3 versus 3.9 +/- 0.8 pmol/l, P < 0.02). 6. In conclusion, fasting concentrations of proinsulin-like molecules are elevated in non-obese subjects with newly diagnosed impaired glucose tolerance. This observation is consistent with defective islet B cell proinsulin processing in this syndrome.

Proopiomelanocortin is the predominant adrenocorticotropin-related peptide in human cerebrospinal fluid.

High mol wt forms of immunoreactive ACTH and beta-endorphin (beta EP) are present in cerebrospinal fluid (CSF). We have quantified these peptides directly in the CSF of 26 patients undergoing routine myelography, using a panel of monoclonal antibody-based two-site immunoradiometric assays, specific for ACTH precursors (both POMC and pro-ACTH cross-react 100%), POMC, ACTH, and beta EP. The mean +/- SD levels of POMC in CSF were 530 +/- 150 pmol/L similar to total ACTH precursor immunoreactivity (414 +/- 83 pmol/L). By comparison, the CSF levels of ACTH (3.2 +/- 0.6 pmol/L) and beta EP (6.7 +/- 2.9 pmol/L) were 100-fold lower. POMC, by virtue of its 1% cross-reactivity in the ACTH immunoradiometric assay, could have also accounted for the ACTH immunoreactivity in CSF. Sephadex G-75 chromatography of CSF confirmed the presence of a single major peak of ACTH precursors eluting at the position of POMC (31K), while ACTH immunoreactivity was not detected at the position of ACTH-(1-39) (4.5K). We also studied the effect of exogenous glucocorticoids on CSF POMC peptides by giving 2.5 mg dexamethasone (0.5 mg, orally, every 6 h for 24 h) to a similar group of age-matched patients before lumbar puncture. No significant differences in CSF peptide content were observed between the two groups. These data suggest that the unprocessed precursor molecule POMC is the predominant peptide of the POMC family in human CSF and should always be considered when interpreting data involving ACTH or other component peptide immunoreactivity in this biological fluid.

Monoclonal antibody-based immunoenzymometric assays of retinol-binding protein

Retinol-binding protein (RBP) is a low-molecular-mass protein (21 kDa), easily filtered in renal glomeruli and very efficiently reabsorbed by the proximal convoluted tubules (PCTs). In PCT dysfunction, high concentrations of RBP are found in urine. Several methods have been used to determine RBP in serum or urine. We describe the production, selection, labeling, and utilization of anti-RBP monoclonal antibodies in two- or one-step immunoenzymometric assays for the determination of RBP. The one-step assay has good precision, with within-run and between-run CVs < 6.6% and 5.9%, respectively. Comparison with radial immunodiffusion (x) showed good agreement: y = 0.068 mg/L + 0.899x (n = 24). Comparison between the one-step (y) and two-step (x) versions of the assay also showed a very good correlation: y = 212 micrograms/L + 0.910x. The one-step assay has been adopted for routine work; it detects transthyretin-bound as well as free RBP and may have clinical usefulness in evaluating the functional status of PCTs.

Free α-Subunit, Free β-Subunit of Human Chorionic Gonadotropin (hCG), and Intact hCG in Sera of Healthy Individuals and Testicular Cancer Patients

To determine the serum concentrations of human chorionic gonadotropin (hCG), its free beta-subunit (hCG beta), and the free alpha-subunit (free alpha) common to all human glycoprotein hormones under physiological and pathological conditions, we developed monoclonal antibody-based immunoenzymometric assays. Free alpha-subunit was detected in the sera of all healthy individuals of both sexes; hCG was measurable in sera of 54% of the men, and 46% were positive for free hCG beta; in nonpregnant women, 69.5% were positive for hCG, 68.4% for the free beta-subunit. Pathological conditions, i.e., hCG-producing tumors, were studied in vitro and in vivo. In vitro, the concentrations of hCG, free hCG beta, and free alpha in tissue-culture supernates of a choriocarcinoma cell-line ("JAR") showed a parallel pattern during time-course analysis. In vivo, in long-term follow-up studies of 13 patients with testicular cancer, serum concentrations of the three analytes paralleled each other, whether the disease was in remission or not. Because of a selective increase of free hCG beta and free alpha in 27% of seminomatous tumor patients and in 13% of the nonseminomatous patients, the percentage of tumor-marker-positive sera was increased from 15% to 42% and 57% to 70%, respectively, by the additional measurement of free hCG beta and free alpha. Thus hCG, free hCG beta, and free alpha are physiologically present in a high percentage of the sera from healthy men, and the determination of free hCG beta and free alpha, although not of prognostic value, improves the diagnostic possibilities in patients with testicular cancer.

Is an immunoassay available for the measurement of bioactive LH in serum?

An in vitro bioassay for luteinizing hormone (LH) is in our opinion the "gold standard" bioassay. The rodent interstitial cell testosterone assay (RICT) is specific for bioactive LH and very sensitive, accurate, and reproducible. Diverse LH standards consistently display parallel dose-response characteristics. Sera also manifest parallel dose-response characteristics throughout reproductive life, with the exception of basal samples from prepubertal children. This indicates that all known hormones with LH bioactivity have a similar bioactive site. The in vivo bioassays for LH used for calibration of World Health Organization standards are more cumbersome and less precise and accurate than the in vitro bioassay. The ovarian ascorbic acid depletion assay corresponds better than the seminal vesicle weight assay with in vitro bioassay. Variation in the ratio of bioactive to immunoreactive LH (B/I) principally reflects variation in LH immunoassay dose-response characteristics, rather than a change in the bioactive moiety of LH. The varying B/I ratio is due to molecular heterogeneity at multiple levels. Different LH standards contain different proportions of nonbioactive but immunoreactive material. The immunoreactive LH isoforms in serum contain different proportions of bioactive material and the isoform distribution differs with reproductive status. Furthermore, the antibodies comprising the various immunoassay systems detect heterogeneous epitopes on LH, which are not necessarily bioactive. B/I ratio disparities indicate lack of specificity of immunoassays for bioactive LH. Polyclonal antibody-based radioimmunoassay requires the use of purified reagents, including a bioactive tracer, in order to achieve high specificity for bioactive LH. The new generation of monoclonal antibody-based immunometric assays yields results that are lower than, but correlate with, LH measured by the in vitro bioassay. The purest of standards, even a recombinant standard, yields results that differ up to 50% or more from one immunoassay to another. Serum LH levels also differ up to two-fold among assays. The immunometric assays have the advantage of being more sensitive and more specific for low levels of LH in serum than radioimmunoassays, but B/I ratio discrepancies remain great. An immunoassay specific for the bioactive "docking site" of human LH isoforms is still needed.

Immunochemical techniques in biological monitoring.

Immunoassays are analytical methods that detect interactions between antibodies and antigens. Immunoassays were used originally to detect large biological molecules. The new generation of these antibody-based assays can detect small synthetic compounds. As a result, immunoassays are being developed specifically for biomarkers of exposure and effect to environmentally prevalent chemicals. Immunochemical detection of parent compounds in blood and tissues, metabolites in excreta, and adducts with DNA and protein have been successfully performed by several investigators. Although there is great potential for use of immunoassays in biological monitoring studies, the limitations of these analyses must be fully understood to prevent improper evaluation of the acquired data. This review will cover some of the background material necessary to understand how an antibody-based assay is developed. The differences between polyclonal and monoclonal antibody-based assays and the importance of antibody class, affinity, specificity, and cross-reactivity must be considered in both study design and data analysis.

Rapid and sensitive detection of Salmonella (0: 6,7) by immunomagnetic monoclonal antibody-based assays

An immunomagnetic technique to detect and identify Salmonella serogroup C1 has been developed. Monoclonal antibodies (MAbs) specific for the O antigen 6,7 of Salmonella lipopolysaccharide (LPS) coupled to magnetic beads were used to isolate the salmonellae. Captured bacteria were easily identified by acridine orange staining and measured by enzyme immunoassays with a conjugate anti-LPS MAb as the detector probe. The whole detection process required 2-3 h and the sensitivity was 10(3)-10(4) bacteria/ml. The presence of blood (10%, v/v) or stool (1%, w/v) components did not interfere with the immunomagnetic assay performance.

Abnormal maternal serum levels of human chorionic gonadotropin free subunits in trisomy 18

We have measured maternal serum levels of free alpha and beta subunits of human chorionic gonadotropin between 8 and 12 weeks of gestation in 704 women at increased risk for trisomy. This group was studied because of advanced maternal age or a previous birth with chromosomal abnormality. All sera had been collected prior to chorion villus biopsy for prenatal diagnosis. Serum levels of free alpha and beta hCG were determined by specific monoclonal antibody-based immunoradiometric assays. Analysis of chorionic tissue showed that in 38 of 704 (5.4%) pregnancies the fetus had a chromosome abnormality. There were 8 fetuses with trisomy 18 (1.1%) and 9 (1.3%) with trisomy 21. In all pregnancies carrying a trisomy 18 fetus, we observed either high levels of free alpha hCG or low levels of free beta hCG or both. More importantly, the calculated ratio of free beta hCG/alpha hCG was less than 0.25 multiples of the median (MoM) in 6 of 8 (75%) trisomy 18 cases. Only 21 of 666 mothers (3.2%) carrying a normal fetus had a ratio less than 0.25 MoM (P less than 0.0001). There was no difference between this ratio in trisomy 21 and normal pregnancy. Thus, when adjusted for gestational age, a low free beta hCG/alpha hCG ratio in maternal serum indicates a pregnancy at high risk [RR = 72 (95% CI 32, 162)] for trisomy 18.

Monoclonal antibody-based plasma assays for fibrin(ogen) and derivatives, and their clinical relevance

The haemostatic balance can basically be described as the equilibrium between fibrin formation (coagulation) and fibrin lysis (fibrinolysis). The status of this balance may therefore be reflected by the products of these two processes. Until recently, the tests for assessment of fibrin(ogen) degradation products were performed in serum since they were based on polyclonal antibodies, which cross-react with fibrinogen. However, the use of serum introduces many artefacts so the utility of these serum tests is limited. New assays have now become available, which can be divided into quantitative enzyme immunoassays (EIAs) and semi-quantitative latex agglutination assays. The new assays can be carried out in plasma since they use highly specific monoclonal antibodies, the majority of which do not cross-react with fibrinogen. This makes it possible to avoid the serum artefacts. Furthermore, these plasma assays can discriminate between degradation products of fibrin and those of fibrinogen (FbDPs and FgDPs, respectively). The possible clinical utility of the new assays is discussed on the basis of literature data on the following clinical states: deep venous thrombosis (DVT) and pulmonary embolism, liver disease and liver transplantation, sickle cell disease, renal diseases, pregnancy and preeclampsia, disseminated intravascular coagulation (DIC), malignancy, coronary artery disease and thrombolytic therapy. Fibrinolysis appears to be accompanied by fibrinogenolysis. Detection of fibrin(ogen) derivatives may be used to rule out DVT and to monitor efficacy of anticoagulant treatment for DVT or DIC, and reflects severity of renal disease but not renal function. High levels of FgDPs were found during orthotopic liver transplantation and thrombolytic therapy. Fibrin(ogen) degradation products cannot be used to predict reperfusion following thrombolytic therapy. The fibrinolytic system remained active during normal and complicated pregnancy and in patients with malignancies. The new assays provide valuable information on fibrin(ogen)olysis in several diseases. More information on the haemostatic balance may be obtained by using these new assays for fibrin(ogen)olysis products in combination with assays for coagulation products.

The Squamous Cell Carcinoma Tumor Marker in Mammary Carcinoma: Comparison of Polyclonal versus Monoclonal Antibody-Based Assays

Pretreatment serum levels of squamous cell carcinoma (SCC)-antigen as determined with a polyclonal and monoclonal antibody-based assay were determined in 136 patients with invasive mammary carcinoma, 8 patients with ductal carcinoma in situ and 23 patients with benign breast diseases. For the polyclonal assay, the overall sensitivity was 44.8% at a cut-off level of 2.0 ng/ml and 25% for 2.5 ng/ml. For the monoclonal assay, the sensitivity was 11.0 and 25.0% at cut-off levels of 2.0 and 1.5 ng/ml, respectively. On the basis of analysis of specificity of the two assays for the benign control group, the cut-off level of the polyclonal assay has to be 2.5 ng/ml in the case of mammary carcinoma, of the monoclonal assay it has to be 2.0 ng/ml. For 175 serum samples of the whole group of patients (pre- and postoperative samples), the polyclonal RIA (x) and the monoclonal IRMA (y) were measured. The orthogonal regression analysis gave the equation: monoclonal IRMA = 1.03 polyclonal RIA - 1.15 ng/ml (Sy.x = 0.48; r = 0.7225). We also came to the conclusion that the SCC-antigen measured by either assay is different.

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins.

Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.

Antibody Responses to Hapten in Thymectomized Mice: Extraordinarily Pronounced Deficiency in IgG1 Production

The effect of thymectomy on the production of antibodies was studied by immunizing mice with hapten-carrier conjugates. Antibody responses were analysed with monoclonal antibody-based quantitative isotype-resolving assays. In spite of bone marrow reconstitution, irradiation without thymectomy caused a long-lasting relative deficiency in responsiveness to T-independent antigens. Even when no visible remnants of the thymus could be observed at the autopsy of thymectomized mice, there appeared to be a gradual recovery of antibody-forming capacity within 4 months, as assessed by the response to a T-dependent antigen. Therefore, some of the thymectomized mice had to be regarded as having recovered with respect to the helper T-cell effect. The antibody responses to T-dependent antigens were improved in all isotypes by a functional T-cell system, but the IgG isotypes seemed to benefit more than IgM. The most conspicuous deficit in antibody production in non-recovered thymectomized mice was observed in the T-dependent responses of the IgG1 isotype (2000-fold reduction in contrast to about 50- to 100-fold in IgG2a, IgG2b, and IgG3).

Foreign Exploration for Predators: A Proposed New Methodology

That most biological control success stories involve parasitic rather than predatory natural enemies is a well-entrenched myth. In fact, recent analyses have shown that introduced parasitoids and predators have not differed significantly in their rates of establishment or success as agents of biological control. However, it is true that introductions of predators have been attempted much less often than introductions of parasitoids. This may be partly because of controversy over the attributes of the ideal natural enemy. A more basic obstacle has been the lack of a methodology for identifying any but the most prey-specific of predators of target pests. I propose a new methodology, using on-the-spot serological stomach analysis of predators. The feasibility of this approach is illustrated with data from monoclonal antibody-based ELISA assays of predators fed larvae of species of the corn earworm complex.

Physiological Studies of Human Chorionic Gonadotropin and Free Subunits in the Amniotic Fluid Compartment Compared to Those in Maternal Serum*

We measured intact hCG, free alpha hCG, and free beta hCG levels in amniotic fluid and maternal serum using specific monoclonal antibody-based immunoradiometric assays. Compared to maternal serum, amniotic fluid had low levels of intact hormone along with high levels of its free subunits. The mean amniotic fluid hCG level was 1 mg/L at 13 weeks, and it progressively decreased to 0.100 mg/L by the 23rd week. Amniotic fluid alpha hCG levels were highest at 15 and 16 weeks (0.340 mg/L) and rapidly declined to concentrations less than 0.050 mg/L at 21 weeks. Amniotic fluid beta hCG concentrations were about 0.200 mg/L between 13 and 16 weeks and progressively decreased until the 23rd week. In vitro stability studies of hCG and its subunits demonstrated that dissociation of hCG into its subunits was not responsible for these results. Both hCG and free subunits detected in amniotic fluid were indistinguishable from standard hCG and free subunit preparations, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our findings are consistent with the hypothesis that the presence of hCG and free subunits in the amniotic fluid represents secretion from the trophoblastic tissue into the amniotic fluid compartment. Furthermore, high serum hCG levels in association with low levels of its free subunits are probably the result of polarized secretion of the intact hormone from the syncytiotrophoblasts into the maternal circulation.