Elimination of Human Chorionic Gonadotropin in Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone in Serum

Abstract

A method to eliminate human chorionic gonadotropin (hCG) in the sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. hTSH in serum containing hCG was reacted with dinitrophenyl monoclonal mouse anti-hTSH β-subunit IgG₁, and the complex formed between the dinitrophenyl IgG₁ and hTSH was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. hCG in the test serum was largely eliminated by washing the polystyrene balls. Subsequently, the complex on the polystyrene balls was reacted with affinity-purified rabbit anti-hCG Fab′-peroxidase conjugate followed by washing. The complex of the dinitrophenyl IgG₁, hTSH and the conjugate was eluted with dinitrophenyl-L-lysine from the polystyrene balls, to which hCG had been nonspecifically adsorbed, and was trapped onto clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. Peroxidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls in the absence and presence of hTSH was not significantly affected by the presence of up to 75,000 IU of hCG per liter of serum. As a result, serum hTSH could be sensitively measured with little interference by hCG.     

Comparison Of Highly Sensitive Sandwich Enzyme Immunoassay And Radioimmunoassay For Human Ferritin

Abstract

A highly sensitive sandwich enzyme immunoassay (EIA) for human ferritin was developed using rabbit anti-ferritin IgG-coated polystyrene balls and affinity-purified rabbit anti-ferritin Fab' labelled with β-D-galactosidase from Escherichia coli and compared with the corresponding sandwich radioimmuno assay (RIA). The specific and nonspecific binding of labelled anti-ferritin to the polystyrene balls were examined in relation to the amount of labelled anti-ferritin used per tube, and the highest sensitivity of each immunoassay (0.2 amol/tube in EIA and 2.5 amol/tube in RIA) was obtained by using the minimal amount of the corresponding labelled anti-ferritin (0.71 fmol in EIA and 4.5 fmol=4436 cpm in RIA) which gave a reliable calibration curve. The sandwich RIA was less sensitive, largely because the specific radioactivity of ¹²⁵I-labelled anti-ferritin used was not sufficiently high.     

A Highly Sensitive Enzyme Immunoassay of Anti-Insulin Antibodies in Guinea Pig Serum

Abstract

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. Insulin bound to anti-insulin antibodies was separated from free insulin by precipitation with polyethylene glycol. Anti-insulin antibodies in the precipitates were dissociated from insulin and inactivated by incubation with 0.1 mol/1 HCl. The amount of insulin dissociated was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab′-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was improved 1,000-fold as compared with that of the enzyme immunoassay previously described, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab′-horseradish peroxidase conjugate.     

A Small Scale Sandwich Enzyme Immunoassay for Macromolecular Antigens Using B-D-Galactosidase from Escherichia Coli and Horseradish Peroxidase as Labels

Abstract

A small scale sandwich enzyme immunoassay for macromolecular antigens using β-D-galactosidase from Escherichia coli and horseradish peroxidase as labels is described. An antibody IgG-coated glass ball of 1.0 mm in diameter was incubated with antigen in 5 μl and subsequently with affinity-purified Fab'-enzyme conjugate in 5 μl. Using B-D-galactosidase, the detection limits of ferritin, IgE, a-fetoprotein and chorionic gonadotropin were 0.001 amol (600 molecules), 0.03 amol, 0.03 amol and 0.07 amol, respectively. Using peroxidase, the detection limits of ferritin, α-fetoprotein and chorionic gonadotropin were 0.005 amol, 0.1 amol and 0.2 amol, respectively. These detection limits were 3 to 30-fold less than those obtained by the previous sandwich enzyme imnunoassays using antibody IgG-coated polystyrene balls of 3.2 mn in diameter in 150 μl.     

Use of Nonspecific F(ab′)2 and Inactive β-D-Galactosidase to Reduce the Nonspecific Binding of Anti-Ferritin Fab′-β-D-Galactosidase Conjugate in Two-Site Enzyme Immunoassay for Ferritin

Abstract

The nonspecific binding of rabbit anti-ferritin Fab′-B-D-galactosidase conjugate to rabbit anti-ferritin IgG-coated polystyrene balls in two-site enzyme immunoassay for ferritin was reduced to various extents by the presence of nonspecific rabbit F(ab′)₂, inactive β-D-galactosidase and related substances with only small decreases in the specific binding, and the detection limit of ferritin was improved up to 10-fold. The use of nonspecific F(ab′)₂, inactive β-D-galactosidase and related substances was suggested to be useful for improving the detection limit of other antigens.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay) for Arginine Vasopressin

Abstract

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 μl of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, ad libitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).     

Highly Specific and Sensitive Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone (hTSH) in Serum Using Monoclonal Anti-hTSH β-Subunit IgG1-Coated Polystyrene Balls and Polyclonal Anti-hTSH β-Subunit Fab′-Horseradish Peroxidase Conjugate

Abstract

A highly specific and sensitive sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. Monoclonal anti-hTSH β-subunit IgG₁?coated polystyrene balls were incubated with serum samples and subsequently with affinity-purified polyclonal anti-hTSH β-subunit Fab′-horseradish peroxidase conjugate. There was no cross-reaction with 38 mIU/tube of human follicle-stimulating hormone, 40 mIU/tube of human luteinizing hormone or 10 IU/tube of human chorionic gonadotropin. The detection limit of hTSH was 0.3 nU/tube or 0.006 mU/1 of serum. Therefore, it was possible to measure very low levels of serum hTSH in pregnant women with Graves' disease. Serum hTSH levels in normal subjects and patients with Graves' disease were 2.45 ± 1.49 (SD) mU/1 (range : 0.61–6.09 mU/1) and 0.086 ± 0.060 (SD) mU/1 (range : < 0.006-0.18 mU/l), respectively. However, the present sandwich enzyme immunoassay was expensive, since affinity-purified polyclonal anti-hTSH β-subunit Fab′-peroxidase conjugate was prepared from rabbit anti-hTSH serum.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay to Measure Peptides by Biotinylation

Abstract

A novel and sensitive noncompetitive enzyme immunoassay for angiotensin I as a peptide model is described. Angiotensin I in buffer containing bovine serum albumin or in plasma was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. the biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. the biotinylated angiotensin I eluted was reacted with anti-angiotensin I Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. the detection limit of angiotensin I was 13 fg (10 amol)/tube and 6.5 ng/1 of plasma, which was 10 to 480-fold lower than that previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. and other peptides could also be measured more sensitively by the present method than by competitive radioimmunoassay.     

Use of Normal IgG and its Fragments to Lower the Non-Specific Binding of Fab'-Enzyme Conjugates in Sandwich Enzyme Immunoassay

Abstract

An antibody IgG-coated polystyrene ball was incubated with an antigen and then with affinity-purified Fab'-enzyme conjugate in the presence of normal IgG, F(ab')₂, Fab' or Fab'-bovine serum albumin conjugate. After washing by incubation at 30[ddot]C for 10 min with shaking, the enzyme activity bound to the polystyrene ball was assayed. The non-specific binding of the Fab'-enzyme conjugate to the polystyrene ball considerably decreased in the presence of normal IgG and the other related proteins, while the specific binding decreased only slightly. As a result, the detection limit of hCG, human IgE and human α-fetoprotein was improved 3 to 10-fold.     

Sensitive Sandwich Enzyme Immunoassay of Human Interleukin-2 Produced In Vitro by Peripheral Blood Mononuclear Cells

Abstract

A sensitive sandwich enzyme immunoassay for human inter-leukin-2 (hIL-2) using monoclonal antibody IS described. A monoclonai anti-hIL-2 IgG-coated poiystyrene ball was incubated with hIL-2 and subsequently with affinity-purified rabbit anti-hIL-2 Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. There was no cross-reaction with interleukins 1α and 1bT, epidermal growth factor, insulin and other protein hormones. The detection limit of hIL-2 was 3 pg/tube or 30 ng/1 using 0.1 ml of cuiture supernatant. When peripheral blood mononuclear cells from healthy subjects aged 22-62 yr were cultured in the absence and presence of phytohemagglutinin P for 48 h, hIL-2 levels in the culture supernatants were <0.03-0.10 μg/1 and 0.18-3.7 μg/1, respectively.     

Improved Sensitive Sandwich Enzyme Immunoassay for Secretory Immunoglobulin a in Human Serum

Abstract

To overcome the IgA interference in enzyme immunoassay for serum secretory IgA (SIgA), a new specific, simple and more sensitive sandwich enzyme immunoassay, fully free of the serum IgA interference, was developed. SIgA standards or samples were added into the wells of polystyrene plate coated with rabbit IgG antibody to human secretory component. After incubation, the wells were washed and then, horseradish peroxidase-labeled Fab′ fragment of goat IgG antibody to human α-chain was added and incubated. The wells were washed again to remove the unbound labeled antibody, and the enzyme activity specifically bound to the well was assayed using 3,3′, 5,5′ - tetramethylbenzidine and H₂O₂ as substrate. The enzyme reaction was stopped by addition of 2M H₂SO₄. The SIgA concentration was determined from the absorbance at 450 nm. The minimun detectable sensitivity was 6ng/ml. The assay system had very good selectivity overcoming the interference of IgA. As the result of high sensitivity, only small amount of sample (2 μ1 for serum) was needed for analysis. In this assay, no cross reactivity was found with purified human IgG, mIgA, IgM or free secretory component (FSC). The recovery of SIgA mixed with human sera or biles was 99.6–108.1%. The coefficients of within-assay and between-assay variation were 5.8–9.3% and 6.2–9.2% respectively. It correlated well with a liquid phase competitive radioimmunoassay for human serum SIgA (r=0.96, n=30, P<0.0l). The level of SIgA in normal human serum was 8.04±3.60 (SD) μg/ml (n=117) and increased significantly in patients with choledocho- lithiasis (57.35±49.70 μg/ml, n=15, P<0.0l). SIgA concentrations in bile samples were also determined by the 2 4′ assay under the condition that FSC did not, interfere with the assay.     

Immune Complex Transfer Enzyme Immunoassay for Anti-Thyroglobulin IgG Using 2,4-Dinitrophenyl-thyroglobulin,Biotinyl-thyroglobulin and Streptavidin-β-D-galactosidase Conjugate

Abstract

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in serum is described. Anti-thyroglobulin IgG in serum was reacted simultaneously with 2,4-dinitrophenyl-thyroglobulin and biotinyl-thyroglobulin. The immune complex formed of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG and, after washing, reacted with streptavidin-β-D-galactosidase conjugate. After washing, the immune complex was eluted from the polystyrene balls with εN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with (anti-human IgG γ-chain) IgG. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Biotinylthyroglobulin and streptavidin-β-D-galactosidase conjugate could be prepared more easily than thyroglobulin-β-D-galactosidase conjugate used in the previous immunoassay. Inactive β-D-galactosidase, used to eliminate interference by anti-β-D-galactosidase antibodies in the previous immunoassay, was not required. The present immunoassay was 300-fold more sensitive than the conventional enzyme immunoassay, although 10-fold less sensitive than the previous immunoassay. Antithyroglobulin IgG was demonstrated in all patients with autoimmune thyroid diseases and 54% of healthy subjects.     

Effect Of Temperature on the Sensitivity of Sandwich Enzyme Immunoassay with Fab'-Horseradish Peroxidase Conjugate

Abstract

Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher at 20°C than at 37°C. The lowest non-specific binding and the highest specific binding were obtained by incubation at 20°C with the maleimide conjugate. As a result, the sensitivity could be more easily improved by incubation at 20°C than at 37°C and the highest sensitivity was obtained with the maleimide conjugate. Similar results were also obtained for other macromolecular antigens such as human ferritin and α-fetoprotein.     

Preparation of Monomeric Affinity-Purified Fab'-ß-D-Galactosidase Conjugate for Immunoenzymometric Assay

Abstract

A Simpler method for the preparation of monomeric affinity-purified Fab'-ß-D-galactosidase conjugate is described. Rabbit (anti-human IgG) serum was subjected to successive processes of pepsin digestion to convert IgG to F(ab')_2′ reduction with 2-mercaptoethy on a column of human IgG-Sepharose 4B. The affinity-purified Fab' thus obtained without using gel filtration was reacted with excess of maleimide groups introduced into ß-D-galactosidase from Escherichia coli. The monomeric Fab'-ß-D-galactosidase conjugate formed was separated from unconjugated Fab' by gel filtration and from unconjugated ß-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B. By immunoenzymometric assay technique for human IgG, the monomeric conjugate was compared with a monomeric conjegate prepared by a previously reported complexmethod and non-monomeric conjugate which contained 3.7 Fab' molecules per ß-D-galactosidase molecule. The present monomeric conjugate provided as sensitive a dose-response curve as the previously reported monomeric conjugate and a more sensitive dose-response curve than the non-monomeric conjugate.     

A Simultaneous Enzyme-Linked Immunoassay for Human Thyroglobulin

Abstract

A “simultaneous” enzyme-linked immunoassay for the measurement of thyroglobulin in human serum was established using polyclonal rabbit antibody, in which rabbit anti-human thyroglobulin IgG-coated silicone rubber rod, rabbit anti-human thyroglobulin monovalent fragment (Fab')-β-D-galactosidase conjugate and serum sample were mixed at the same time. In order to improve the assay performance, an appropriate amount of Fab'-β-D-galactosidase conjugate was carefully chosen.

The present assay was simple, rapid, highly sensitive and excellently reproducible. The sensitivity of the assay was approximately 1.5 amol/tube corresponding to 0.5 ng/ml using 2 μ1 of serum, which was higher than that in the two-step sandwich enzyme-linked immunoassay previously reported. The coefficients of variation for different levels of serum thyroglobulin determined by the present assay ranged from 3.4–7.2% and 2.5–6.1 % for within and between run, respectively.     

Simpler and More Sensitive Immune Complex Transfer Enzyme Immunoassay for Anti-Htlv-I Igg Using Modified Polystyrene Beads,Microplates and Fluororeader

Abstract

In the previous immune complex transfer enzyme immunoassay for anti-HTLV-I IgG, the transfers of polystyrene beads in and out of test tube were handled with tweezers, and the bound β-D-galactosidase activity was measured with a fluorometer. The use of tweezers was considered the primary cause of false-positivity by carryover. Furthermore, the testing of many samples using a tweezer as a means of transfer was difficult. In the present immune complex transfer enzyme immunoassay, polystyrene beads were attached to plates through cylindrical bars and were used in microplate wells. Therefore, no tweezers were required. The bound β-D-galactosidase activity was measured with a fluororeader. This allowed the elimination of false-positivity due to carryover and made it easier to test many samples with higher sensitivity and reliability.     

A Highly Sensitive Sandwich Enzyme Immunoassay for Insulin in Human Serum Developed Using Capybara Anti-Insulin Fab' -Horseradish Peroxidase Conjugate

Abstract

A highly sensitive sandwich enzyme immunoassay for insulin in human serum has been developed using capybara anti-insulin serum. Capybara anti-insulin IgG-coated polystyrene balls were incubated with serum samples in the presence of 0.4 M NaCl and then with capybara anti-insulin Fab'-horseradish peroxidase conjugate.

The peroxidase activity bound to the polystyrene balls was correlated to the amount of insulin to be assayed. Serum interference was eliminated by the presence of 0.4 M NaC1, and there was no need to add insulin-free serum to a standard curve. The sensitivity was 4 nU/tube or 0.2 μU/ml of serum when 20 μ1 of serum samples was used. The recovery of insulin added to human serum was 92–97 %. The coefficients of within-assay and between-assay variations were 5.1–7.2 % and 7.6–9.2 %, respectively. The regression equation and correlation coefficient to radioimmunoassay were Y(EIA)=0.91×(RIA)-2.4 and 0.97 (n=76), respectively.     

A Micro-Scale Preparation of Affinity-Purified Fab'-Enzyme Conjugates with High Purity for Enzyme Immunoassay

Abstract

A micro-scale method was developed for conjugating a small amount of affinity-purified Fab' to enzymes through thiol groups in the hinge of Fab'. 2,4-Dinitrophenyl groups were introduced into rabbit anti-hCG F(ab')₂ before affinity-purification, and the 2,4-dinitrophenyl F(ab')₂ (0.2–2.0 mg) was affinity-purified by elution from a column of hCG-Sepharose 46 with 0.1 M glycine-HC1 buffer, pH 2.5, containing normal rabbit F(ab')₂ (1.0 mg) as a carrier. The affinity-purified 2,4-dinitrophenyl F(ab')2 mixed with normal F(ab')₂ was split into Fab' by reduction, reacted with maleimide groups introduced into enzymes and subjected to gel filtration to separate the conjugates from unconjugated components. Finally, the affinity-purified 2,4-dinitrophenyl anti-hCG Fab'-enzyme conjugates were separated from normal Fab'-enzyme conjugates and unconjugated enzyme, if any, by affinity chromatography on a column of (anti-2,4-dinitro-phenyl) IgG-Sepharose 4B. The conjugate preparations obtained by the micro-scale method were satisfactory in purity, antigen-binding activity and usefulness for sandwich enzyme immunoassay.

This method is applicable to the conjugation not only with horseradish peroxidase but with other enzymes, while a previously reported method is applicable only to the conjugation with horseradish peroxidase.     

Highly Sensitive Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone (hTSH) in Serum Using Monoclonal Anti-hTSH β-Subunit IgG1-Coated Polystyrene Balls and Polyclonal Anti-Human Chorinic Gonadotropin Fab′-Horseradish Peroxidase Conjugate

Abstract

A highly sensitive sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. Monoclonal anti-hTSH β-subunit IgG₁-coated polystyrene balls were incubated with serum samples and subsequently with polyclonal anti-human chorionic gonadotropin Fab¹-horseradish peroxidase conjugate. Neither hTSH nor anti-hTSH serum was required except for a small amount of hTSH as a standard. The detection limit of hTSH was 0.1 nU/tube or 0.005 mU/1 for most serum samples except for those from pregnant women, since there was no cross-reaction with 209 IU/1 of human follicle-stimulating hormone and 439 IU/1 of human luteinizing hormone when 20 μ1 of serum sample was used. Serum hTSH levels in normal subjects and Graves¹ disease were 1.22 ± 0.70 (SD) mU/1 (range: 0.26–2.65 mU/1) and 0.080 ± 0.062 (SD) mU/1 (range: < 0.005–0.17 mU/1), respectively.