Thyroid Stimulating Hormone Immunoassay Research Articles

Quantitative Detection of Thyroid-Stimulating Hormone in Patient Samples with a Nanomechanical Single-Antibody Spectro-Immunoassay.

Functional disorders of the thyroid remain a global challenge and have profound impacts on human health. Serving as the barometer for thyroid function, thyroid-stimulating hormone (TSH) is considered the single most useful test of thyroid function. However, the prevailing TSH immunoassays rely on two types of antibodies in a sandwich format. The requirement of repeated incubation and washing further complicates the issue, making it unable to meet the requirements of the shifting public health landscape that demands rapid, sensitive, and low-cost TSH tests. Herein, a systematic study is performed to investigate the clinical translational potential of a single antibody-based biosensing platform for the TSH test. The biosensing platform leverages Raman spectral variations induced by the interaction between a TSH antigen and a Raman molecule-conjugated TSH antibody. In conjunction with machine learning, it allows TSH concentrations in various patient samples to be predicted with high accuracy and precision, which is robust against substrate-to-substrate, intra-substrate, and day-to-day variations. It is envisioned that the simplicity and generalizability of this single-antibody immunoassay coupled with the demonstrated performance in patient samples pave the way for it to be widely applied in clinical settings for low-cost detection of hormones, other molecular biomarkers, DNA, RNA, and pathogens.

How low can we (reliably) go? A method comparison of thyroid-stimulating hormone assays with a focus on low concentrations.

International guidelines concerning subclinical hyperthyroidism and thyroid cancer advice absolute cut-off values for aiding clinical decisions in the low range of thyroid-stimulating hormone (TSH) concentrations. As TSH assays are known to be poorly standardized in the normal to high range, we performed a TSH assay method comparison focusing on the low range. Sixty samples, selected to cover a wide range of TSH concentrations (<0.01 to 120 mIU/L) with oversampling in the lower range (<0.4 mIU/L), were used for the method comparison between three TSH immunoassays (Cobas, Alinity and Atellica). In addition, 20 samples were used to assess the coefficient of variation from duplicate measurements in these three methods. The TSH immunoassays showed standardization differences with a bias of 7-16% for the total range and 1-14% for the low range. This could lead to a different classification of 1.5% of all measured TSH concentrations <0.40 mIU/L measured in our laboratory over the last 6 months, regarding the clinically important cut-off value of TSH = 0.1 mIU/L. As the imprecision of the immunoassays varied from 1.6-5.5%, this could lead to a similar reclassification as the bias between immunoassays. We established the standardization differences of frequently used TSH assays for the total and low concentration ranges. Based on the proportional bias and the imprecision, this effect seems to have limited clinical consequences for the low TSH concentration range. Nevertheless, as guidelines mention absolute TSH values to guide clinical decision-making, caution must be applied when interpreting values close to these cut-offs.

Instability of harmonized thyroid-stimulating hormone immunoassays in clinical practice.

Thyroid-stimulating hormone (TSH) harmonization is effective in minimizing differences between the results of immunoassays in healthy subjects. However, the effectiveness of TSH harmonization in clinical practice has not been investigated. The aim of this study was to evaluate the instability of TSH harmonization in clinical practice. We compared the reactivities of four harmonized TSH immunoassays using combined difference plots of 431 patients. We selected patients with statistically significant deviations in TSH levels and analyzed their thyroid hormone levels and clinical characteristics. The combined difference plots showed that one harmonized TSH immunoassay exhibited markedly different reactivity even after TSH harmonization compared with the other three immunoassays. Among 109 patients with mild-to-moderate elevation of TSH levels, we selected 15 patients with statistically significant deviations in TSH levels according to the difference plots of three harmonized TSH immunoassays, excluding one immunoassay that showed different reactivity. The thyroid hormone levels of three patients were misclassified as hypothyroidism or normal due to deviating TSH levels. In terms of clinical characteristics, these patients were in poor nutritional status and general condition, possibly due to their severe illness (e.g., advanced metastatic cancer). We have confirmed that TSH harmonization in clinical practice is relatively stable. However, some patients showed deviating TSH levels in the harmonized TSH immunoassays, indicating the need for caution, particularly in poorly nourished patients. This finding suggests the presence of factors that contribute to the instability of TSH harmonization in such cases. Further investigation is warranted to validate these results.

Correlation of thyroid hormone measurements with thyroid stimulating hormone stimulation test results in radioiodine-treated cats.

BackgroundIatrogenic hypothyroidism can develop after radioiodine‐I131 (RAI) treatment of hyperthyroid cats and can be diagnosed using the thyroid stimulating hormone (TSH) stimulation test.ObjectivesTo assess the effect of noncritical illness on TSH stimulation test results in euthyroid and RAI‐treated cats. To assess the correlation of low total‐thyroxine (tT4), low free‐thyroxine (fT4), and high TSH concentrations with TSH stimulation test results.AnimalsThirty‐three euthyroid adult cats and 118 client‐owned cats previously treated with RAI.MethodsTotal‐thyroxine, fT4, and TSH were measured, and a TSH stimulation test was performed in all cats. Euthyroid control cats were divided into apparently healthy and noncritical illness groups. RAI‐treated cats were divided into RAI‐hypothyroid (after‐stimulation tT4 ≤ 1.5 μg/dL), RAI‐euthyroid (after‐stimulation tT4 ≥ 2.3 μg/dL OR after‐stimulation tT4 1.5‐2.3 μg/dL and before : after tT4 ratio > 1.5), and RAI‐equivocal (after stimulation tT4 1.5‐2.3 μg/dL and tT4 ratio < 1.5) groups.ResultsNoncritical illness did not significantly affect the tT4 following TSH stimulation in euthyroid (P = .38) or RAI‐treated cats (P = .54). There were 21 cats in the RAI‐equivocal group. Twenty‐two (85%) RAI‐hypothyroid cats (n = 26) and 10/71 (14%) of RAI‐euthyroid cats had high TSH (≥0.3 ng/mL). Twenty‐three (88%) RAI‐hypothyroid cats had low fT4 (<0.70 ng/dL). Of the 5 (7%) RAI‐euthyroid cats with low fT4, only one also had high TSH. Only 5/26 (19%) RAI‐hypothyroid cats had tT4 below the laboratory reference interval (<0.78 μg/dL).Conclusions and Clinical RelevanceThe veterinary‐specific chemiluminescent fT4 immunoassay and canine‐specific TSH immunoassay can be used to aid in the diagnosis of iatrogenic hypothyroidism in cats.

Commercially Bottled Purified Water as an Alternative Instrument Feed Water in Automated Time-Resolved Fluorescent Immunoassay for TSH, 17-OHP and IRT in Neonatal Screening

Objective. The study was undertaken to determine if commercially bottled purified water can be used as substitute instrument feed water for three (3) newborn screening immunoassays. Methdology. A total of 294 control samples and 300 patient samples were included in this study. Accuracy and precision studies using control samples, and parallel testing using patient samples, were done to compare the use of clinical laboratory reagent water (CLRW) and commercially bottled purified water (CBPW) in the performance of automated time-resolved fluorescent immunoassay of thyroid stimulating hormone (TSH), 17α-OH-progesterone (17-OHP) and immunoreactive trypsinogen (IRT). Results. The use of CBPW as instrument feed water for measurements of TSH, 17-OHP and IRT levels by automated time-resolved fluorescent immunoassay using AutoDELFIA (Perkin-Elmer) in NBS has an acceptable accuracy and precision compared to using CLRW. The parallel testing using patient samples showed that, overall, the performance of using CBPW in automated time-resolved fluorescent immunoassay for TSH, 17-OHP, and IRT is acceptable, compared with using CLRW as instrument feed water. Conclusion. Commercially bottled purified water can be used as substitute when setting up a laboratory water purification system is too expensive for a laboratory, or as back up to clinical laboratory reagent water when there is breakdown of the installed water purification system to be used as instrument feed water in automated time-resolved fluorescent immunoassay of TSH, 17-OHP and IRT in NBS using AutoDELFIA (Perkin-Elmer).

A multicenter study for the evaluation of the reference interval for TSH in Italy (ELAS TSH Italian Study)

Background The aims of this study were: (1) to calculate reliable thyroid stimulating hormone (TSH) reference intervals using laboratory databases; (2) to evaluate the relationship between TSH, sex and age values in different large Italian populations. Methods The TSH values stored in the laboratory information system of clinical laboratories of four Italian city hospitals, including 146,801 TSH measurements (with the respective age and sex data of individuals) were taken in consideration. Assuming a log-normal distribution, to log-transformed TSH values were applied the Dixon's iterative principle in order to exclude the outliers. At the end of this iterative process 142,821 log-transformed TSH results remained. The four clinical laboratories measured serum TSH concentrations using the same TSH immunoassay method (Access TSH 3rd IS, using UniCel DxI platform). Results The TSH reference interval calculated in the present study (0.362-5.280 mIU/L) is similar to that suggested by the manufacturer for the Access TSH 3rd IS assay (0.45-5.33 mIU/L). TSH values in females were significantly higher than in males (females: mean=2.06 mIU/L; standard deviation [SD]=1.26 mIU/L; n=101,243; males: mean=1.92 mIU/L; SD=1.19 mIU/L; n=41,578; p<0.0001). Moreover, a negative linear relationship was observed between TSH throughout all interval age values (from 0 to 105 years). Conclusions The results of the present multicenter study confirm that data mining techniques can be used to calculate clinically useful reference intervals for TSH. From a pathophysiological point of view, our results suggest that some Northern populations of Italy might still suffer some harmful effects on the thyroid gland due to mild to moderate iodine intake deficiency. Specific clinical trials are needed to confirm these results.

Upconversion Cross-Correlation Spectroscopy of a Sandwich Immunoassay.

Fluorescence correlation and cross‐correlation spectroscopy (FCS/FCCS) have enabled biologists to study processes of transport, binding, and enzymatic reactions in living cells. However, applying FCS and FCCS to samples such as whole blood and plasma is complicated as the fluorescence bursts of diffusing labels can be swamped by strong autofluorescence. Here we present cross‐correlation spectroscopy based on two upconversion nanoparticles emitting at different wavelengths on the anti‐Stokes side of a single excitation laser. This upconversion cross‐correlation spectroscopy (UCCS) approach allows us to completely remove all Stokes shifted autofluorescence background in biological material such as plasma. As a proof of concept, we evaluate the applicability of UCCS to a homogeneous sandwich immunoassay for thyroid stimulating hormone measured in buffer solution and in plasma.

Improving the sensitivity of immunoassays by reducing non-specific binding of poly(acrylic acid) coated upconverting nanoparticles by adding free poly(acrylic acid).

Upconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF4:Yb3+,Er3+ type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540nm upon 980nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1ng·L-1 to 0.48ng·L-1 in case of cTnI and from 0.070 mIU·L-1 to 0.020 mIU·L-1 in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings. Graphical abstract The presence offree poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific proteinbinding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctlymore sensitive assaysto beperformed.

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L−1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates.Graphical We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Heterogeneous time-resolved electrochemiluminoimmunoassay of thyroid stimulating hormone with magnetic beads at oxide-covered aluminum electrode

The heterogeneous immunoassay of thyroid stimulating hormone (TSH) was detected by the time-resolved cathodic electrochemiluminescence immunoassay (tr-CECLIA) method using magnetic beads as a mobile support. The magnetic beads coated with sandwich type TSH immunoassay were captured with a magnet for washing and detection processes. The time-resolved cathodic electrochemiluminescence (tr-CECL) signal of Tb(III) chelate label was generated by cathodic pulse polarization in the aluminum working electrode and platinum counter electrode system. The detection method causes injection of high energy electrons into the aqueous solution near the aluminum electrode and creates rigid simultaneous oxidative and reductive conditions that excitate the Tb(III) chelate used as a label luminophore in the heterogeneous sandwich type immunoassay of TSH in the surface of the magnetic beads. The limit of detection of the method was about 50mIUL−1. The precision of it was noticed to be good; the coefficient variation percentage was realized to be lower than 10 %. Unfortunately the limit of detection is not good enough for determination of analyte levels of very low concentrations for instance TSH in body fluids. The possible application areas of the method are in highly sophisticated micro or nano fluidic detection and sensor systems where the aspiration level of the analyte detection limit is not very high and the mobility and manageability and the large surface area of the magnetic beads can be utilized efficiently in separating, washing, moving, coating and detecting processes. In the case of tr-CECLIA the presented method makes possible to use multipurpose working electrodes instead of disposable ones.

Harmonization protocols for TSH immunoassays: a multicenter study in Italy.

Systematic difference between thyroid-stimulating hormone (TSH) immunoassays may produce misleading interpretation when samples of the same patients are measured with different methods. The study aims were to evaluate whether systematic differences are present among TSH immunoassays, and whether it is possible to obtain a better harmonization among TSH methods using results obtained in external quality assessment (EQA) schemes. Seven Italian clinical laboratories measured TSH in 745 serum samples of healthy subjects and patients with thyroid disorders. These samples were also re-measured by two reference laboratories of the study with the six TSH immunoassays most popular in Italy after 2 months of storage at -80 °C. Moreover, these data were compared to 53,823 TSH measurements, obtained by laboratories participant to 2012-2015 EQA annual cycles in 72 quality control samples (TSH concentrations from about 0.1 mIU/L to 18.0 mIU/L). TSH concentrations were recalibrated using a mathematical approach based on the principal component analysis (PCA). Systematic differences were found between the most popular commercially available TSH immunoassays. TSH concentrations measured by the clinical laboratories were very closely correlated to those measured with the same method by reference laboratories after 2 months of storage at -80 °C. After recalibration using the PCA approach the variation of TSH values significantly decreased from a median pre-calibration value of 13.53% (10.79%-16.53%) to 9.63% (6.90%-13.21%) after recalibration. Our data suggest that EQA schemes are useful to improve harmonization among TSH immunoassays and also to produce some mathematical formulas, which can be used by clinicians to better compare TSH values measured with different methods.

Harmonization protocols for thyroid stimulating hormone (TSH) immunoassays: different approaches based on the consensus mean value

The lack of interchangeable laboratory results and consensus in current practices has underpinned greater attention to standardization and harmonization projects. In the area of method standardization and harmonization, there is considerable debate about how best to achieve comparability of measurement for immunoassays, and in particular heterogeneous proteins. The term standardization should be used only when comparable results among measurement procedures are based on calibration traceability to the International System of Units (SI unit) using a reference measurement procedure (RMP). Recently, it has been promoted the harmonization of methods for many immunoassays, and in particular for thyreotropin (TSH), as accepted RMPs are not available. In a recent paper published in this journal, a group of well-recognized authors used a complex statistical approach in order to reduce variability between the results observed with the 14 TSH immunoassay methods tested in their study. Here we provide data demonstrating that data from an external quality assessment (EQA) study allow similar results to those obtained using the reported statistical approach.

Affinity assisted selection of antibodies for Point of Care TSH immunoassay with limited wash

BackgroundMolecular binding characteristics of several thyroid stimulating hormone (TSH) antibodies were determined for the TSH antigen, along with its closely related endogenous interfering hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). MethodsThis data was compared to the same antibodies used in the low wash sandwich ELISA immunoassay system, the Point of Care i-STAT® immunoassay. From this information we developed binding criteria useful in the low wash i-STAT® immunoassay to permit good signal generation from TSH and low cross-reactivity from its interfering hormones. For the TSH Assay we have developed characteristics that enable antibody selection in the i-STAT® immunoassay cartridge. Our antibody screening approach used a dot blot approach as a first screen to select for the most useful antibodies. We then compared a FRET (Förster Resonance Energy Transfer) and electrochemical cartridge approach to determine the appropriate antibody combinations. ResultsBoth methods generated similar data, but the FRET method was not capable of differentiating the antibody with the best characteristics as a capture antibody or a detection conjugate in a sandwich ELISA assay. Finally, we performed binding characterizations of the antibodies using each of the above mentioned glycoproteins. ConclusionsWe found that we need sub-picomolar detection of TSH, and at least 100 fold or higher values for the cross-reacting species.

Cell-phone-based measurement of TSH using Mie scatter optimized lateral flow assays

Semi-quantitative thyroid stimulating hormone (TSH) lateral flow immunochromatographic assays (LFA) are used to screen for serum TSH concentration >5mIUL−1 (hypothyroidism). The LFA format, however, is unable to measure TSH in the normal range or detect suppressed levels of TSH (<0.4mIUL−1; hyperthyroidism). In fact, it does not provide quantitative TSH values at all. Obtaining quantitative TSH results, especially in the low concentration range, has until now required the use of centralized clinical laboratories which require specimen transport, specialized equipment and personnel, and result in increased cost and delays in the timely reporting of important clinical results. We have conducted a series of experiments to develop and validate an optical system and image analysis algorithm based upon a cell phone platform. It is able to provide point-of-care quantitative TSH results with a high level of sensitivity and reproducibility comparable to that of a clinical laboratory-based third-generation TSH immunoassay. Our research approach uses the methodology of the optimized Rayleigh/Mie scatter detection by taking into consideration the optical characteristics of a nitrocellulose membrane and gold nanoparticles on an LFA for quantifying TSH levels. Using a miniature spectrometer, LED light source, and optical fibers on a rotating benchtop apparatus, the light intensity from different angles of incident light and angles of detection to the LFA were measured. The optimum angles were found that the minimized Mie scattering from nitrocellulose membrane, consequently maximizes the Rayleigh scatter detection from the gold nanoparticles in the LFA bands. Using the results from the benchtop apparatus, a cell-phone-based apparatus was designed which utilized the embedded flash in the cell phone camera as the light source, piped the light with an optical fiber from the flash through a collimating lens to illuminate the LFA. Quantification of TSH was performed in an iOS application directly on the phone and verified using the code written in MATLAB. The limit of detection of the system was determined to be 0.31mIUL−1 (never achieved before on an LFA format), below the commonly accepted minimum concentration of 0.4mIUL−1 indicating clinical significance of hyperthyroidism. The system was further evaluated using human serum showing an accurate and reproducible platform for rapid and point-of-care quantification of TSH using a cell phone.

Functional sensitivity of seven automated thyroid stimulating hormone immunoassays

BackgroundSerum thyroid stimulating hormone (TSH) measurements are useful for detecting clinical and subclinical primary hypo- and hyperthyroidism in ambulatory patients. For diagnosis of hyperthyroidism, the functional sensitivity (FS) is an important performance criterion, and current guidelines recommend an FS of ≤0.02m IU/l for “third” generation performance. MethodsWe evaluated TSH FS for the Access 2, Advia Centaur, Architect i2000, Dimension ExL Modular Analytics E170, Immulite 2000 and Dimension Vista 1500 automated immunoassays using serum pools tested over a 6week period using 2 reagent lots and 2 calibrations. FS was determined by fitting a power function to the imprecision data using KaleidaGraph software. ResultsThe FS (m IU/l) for Access 2, ADVIA Centaur, ARCHITECT i2000, Dimension ExL, Modular Analytics E170, Immulite 2000, and Dimension Vista 1500 systems were determined to be 0.039, 0.006, 0.007, 0.003, 0.008, 0.003, and 0.003, respectively. The lowest and next to lowest pools had overall mean TSH concentrations of 0.012m IU/l and 0.020m IU/l, respectively and a range of concentrations of 0.005 to 0.022m IU/l and 0.007 to 0.077m IU/l, respectively. ConclusionsAll assays showed excellent performance in FS consistent with a “third” generation claim except for the Access 2 system. Further harmonization of TSH immunoassays is required, especially at lower concentrations.

Development of a highly sensitive magnetic particle-based chemiluminescence enzyme immunoassay for thyroid stimulating hormone and comparison with two other immunoassays

Development of a highly sensitive magnetic particle-based chemiluminescence enzyme immunoassay for thyroid stimulating hormone and comparison with two other immunoassays

Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well's bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu-biotin binding densities of 0.080 and 0.47 pmol/mm(2), respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay.

Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone

A highly sensitive “two-site” chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin–streptavidin system (BSAS). The HRP–luminol–H 2O 2 chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP–streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1–40 mU L −1. Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L −1 which was 10-fold higher than the HRP–MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.

Conjugates of the Ca2+ -regulated photoprotein obelin with immunoglobulins: synthesis and use as labels in bioluminescent immunoassay

An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 microIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).

Bispecific-Ab-based immunoassay of thyroid-stimulating hormone.

A bispecific F(ab')2 fragment recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (AP) was prepared by disulfide bond exchange between F(ab')2 fragments of IgG1 mAb against TSH and AP. AP was polymerized by glutaraldehyde, and a sandwich enzyme-linked immunosorbent assay for TSH was developed by using the AP polymers and the bispecific F(ab')2 fragment. In this assay, the preparation of covalently linked AP-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced. The sensitivity for TSH increased in proportion to the degree of AP polymerization, and the lower detection limit obtained with the AP trimer was 0.5 muU/ml. The use of the bispecific F(ab')2 fragment allows us to use monomers and polymers of AP and thereby regulates the sensitivity of the assay.