Abstract: A reagent for use in a "sandwich" enzyme-immunoassay comprising a polymer of a bioactive substance-specific antibody:enzyme conjugate, wherein said antibody is conjugated to said enzyme by means of heterobifunctional cross-linking agent and a "sandwich" enzyme-immunoassay employing the polymer.

Abstract: A method for the quantification of an antigen, antibody, or antigen-antibody complex in a sample solution involving measuring the results of an immunochemical agglutination reaction or an agglutination inhibition reaction by spectrophotometry, wherein after the initiation of the agglutination between the antigen, antibody, or antigen-antibody complex to be determined and sensitized carrier particles to which a substance specifically bindable to the said antigen, antibody, or antigen-antibody complex is bound, a fixing compound is added to fix aggregates formed by the agglutination. A measuring reagent kit or pack includes all the components for use in carrying out the above method.

Abstract: A substituted aldosterone of the formula: ##STR1## wherein either one of R.sup.1 and R.sup.2 is hydrogen and the other is --S(CH.sub.2).sub.m COR.sup.3 or --OCO(CH.sub.2).sub.n COR.sup.3, provided that when R.sup.1 is hydrogen, R.sup.2 is --S(CH.sub.2).sub.m COR.sup.3 or --OCO(CH.sub.2).sub.n COR.sup.3 and when R.sup.2 is hydrogen, R.sup.1 is --S(CH.sub.2).sub.m COR.sup.3 ; m being an integer from 1 to 3, n being an integer from 1 to 5 and R.sup.3 being hydroxyl, lower alkoxy or a residue of tyramine, tyrosine lower alkyl ester, histamine, histidine, 7-aminoheptanoyltyrosine lower alkyl ester or .beta.-D-galactosidase as optionally iodinated, or its (18-20)-acetal 20,21-ketonide, which is useful as the reagent in determination of aldosterones by radioimmunoassay or enzyme immunoassay.

Abstract: The present invention relates to novel peptide compounds, of which the chemical structure is related to that of the polypeptide hormone having a thymic activity, isolated from the blood serum of pig, and the application of these novel compounds for therapeutic purposes. This invention also relates to intermediates useful in preparing the active compounds of the invention.

Abstract: Compounds useful in a simultaneous multiple assay for analytes such as steroids, proteins, peptides, carbohydrates or drugs. The compound or compounds are prepared by labelling an individual analyte with a radioisotope through a chelating agent to form a coordinated compound. The assay uses one or more chelated labelled analytes with one or more labelled analytes wherein each radioisotope is different.

Abstract: A homogeneous specific binding assay device, a method for its preparation, and a method for its use in determining a ligand, such as antigen, hapten, or antibody, in, or the ligand binding capacity of, a liquid sample. The test device comprises a solid carrier member, such as a fibrous web matrix, e.g., paper, or a polymeric film or gel, incorporated with reagents for a homogeneous specific binding assay system which produces a detectable response, usually an electromagnetic radiation signal, that is a function of the presence or amount of the ligand in or the ligand binding capacity of the sample. Useful homogeneous specific binding assay systems include those involving enzyme substrate labels, enzyme prosthetic group labels, and enzyme labels. The detectable response preferably is a luminescent, fluorescent, spectrophotometric, or colorimetric response, which is measurable by visual observation or instrument means.

Abstract: The disclosure relates to an in vitro diagnostic method of screening for immune deficiency in a nephrotic patient comprising administering the presence or absence of the lymphokine, soluble immune response suppressor, in a urine sample of said patient.

Abstract: The present invention relates to an estrogen complex which consists of an estrogen or an estrogen derivative complexed with an organometallic compound containing at least one free carbonyl ligand, the said estrogen complex containing at least one free hydroxyl radical and no free phenolic hydroxyl radical in the .alpha.-position to the site where the organometallic compound is attached.The complexes according to the invention are useful for the determination of hormone receptors.

Abstract: There are provided bifunctional chelating agents, which are analogues of EDTA, conjugates of same with a variety of haptens and conjugates of such conjugates with haptens with certain metals, forming metal complexes. The metal complexes have a variety of uses, among these various assays for the determination of haptens and macromolecules. There is also provided a process for the production of the above. There are provided radioimmunoassays based on the above conjugates with metal cations.

Abstract: A biochemical method of assaying for ligand molecules in fluids based on the specific interaction of a ligand and a ligand-recognition molecule that binds the ligand wherein a free radical forming group is covalently coupled to label either a ligand sought to be assayed or the ligand recognition molecule or is internally generated from the ligand or ligand recognition molecule, and directly or indirectly assaying for the ligand.

Abstract: A method of measuring a biological ligand comprises allowing to coexist a biologically active composition comprising an immobilization phase of a particular antibody or a particular ligand and an immobilization phase of a biotinyl enzyme or a biotinyl enzyme inhibitor, a water-soluble conjugate of the ligand or the antibody and the biotinyl enzyme inhibitor or the biotinyl enzyme, and the ligand to be measured in an aqueous solution, and measuring the remaining biotinyl enzyme activity or the biotinyl enzyme inhibitory activity. This method is highly sensitive and specific, and it is suitable as a clinical test for the determination of physiological substances and trace components in humoral fluid.

Abstract: The present invention relates to a simple and accurate method to be applied in immunoassay technique for separating the free ligand from the antibody-bound ligand.The method involves the use of a solvent extraction operation which at equilibrium provides the formation of two distinct phases the free- and bound-fraction. The solvent to be utilized should be slightly water miscible or completely water immiscible, having the property of marked selective extraction power toward the gamma-labelled constituent to be determined. The method is applicable in a liquid-liquid system, a solid-liquid system and solid-solid system.The method is useful for radioimmunoassay, free radical assay or metallo-immunoassay, being most versatile having also the advantage that as a result of the free ligand being extracted into the solvent, the determination of the quantity of gamma-labelled substance in the free and/or bound fraction compares very favorably with the known prior art methods in immunoassays.

Abstract: An immunoassay is provided for cholesterol epoxide. To prepare the antibodies used in the immunoassay, novel immunogens, are prepared which comprise a 3,5(6)-transdiaxial-dihydroxycholestane-6(5)-yl-hapten adduct linked to a covalently bonded bridge to a carrier protein. To detect cholesterol epoxide in the sample, it is converted to the hapten adduct, then contacted with the selected antibody.

Abstract: A process of carrying out a specific binding assay, for the qualitative detection or quantitative or semi-quantitative determination of an analyte which forms a component of a specific binding reaction, using a labelled conjugate form of a derivative or analogue of one of the relevant specific binding partners, said labelled form being capable of emitting delayed luminescence upon photo-excitation, characterized in that the labelled conjugate form of one of the relevant specific binding partners comprises a derivative or analogue of the specific binding partner with a derivative of a luminescent substance which shows a delayed light emission upon photoexcitation, the delayed light emission of which is subject to quenching by molecular oxygen, and characterized further in that the assay is carried out by the use of a time-resolved photometric method in the presence of an effective amount of a quench-inhibiting substance able to prevent quenching by molecular oxygen.

Abstract: Monoclonal antibodies specific for thyroxine (T.sub.4) are produced by two new and separate hybridoma cell lines. Combinations of the monoclonal antibodies from the two cell lines are used in an immunoassay for T.sub.4 of high accuracy over the range of T.sub.4 concentrations encountered in serum samples.

Abstract: A process for determining the presence of polyvalent antigens by incubation with three receptors is presented. In addition, a kit for carrying out this process is provided as well. One of said receptors is bound to a solid support and the other two, in solution, derive from the same animal species.

Abstract: The invention relates to a method and novel reagents for detecting a ligand in a biological sample. In the method, the sample is combined with (i) an enzyme or an enzyme bound to a first macromolecular compound, (ii) substrate for the enzyme, and (iii) an antibody complex comprising anti-ligand bound to anti-enzyme and a second macromolecular compound bound to either one or both of the anti-ligand and anti-enzyme. The activity of the enzyme varies as a function of the unknown amount of ligand contained in the biological sample.

Abstract: A phenolic compound is used to enhance the sensitivity of a luminescent reaction such as carried out in an immunoassay between a peroxidase enzyme, an oxidant, and a chemiluminescent 2,3-dihydro-1,4-phthalazinedione. Preferably, the phenolic compound is 4-iodophenol, 4-phenylphenol or 2-chloro-4-phenylphenol. In the preferred embodiment, horseradish peroxidase is coupled to an antibody to the substance to be assayed.

Abstract: Analogs of rCRF and oCRF are disclosed that can be administered to achieve a substantial elevation of ACTH, .beta.-endorphin, .beta.-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone levels and/or a lowering of blood pressure over an extended period of time. One analog which has been found to be particularly potent is: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met- Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu -Leu-Glu-Glu-Ala-NH.sub.2. In the analogs, one or more of the first five N-terminal residues may be deleted or may be substituted by a peptide up to 10 amino acids long and/or by an acylating agent containing up to 7 carbon atoms. A number of other substitutions may also be made throughout the chain. These analogs or pharmaceutically or veterinarily acceptable salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier, can be administered to mammals, including humans.

Abstract: Homogeneous assay for determining thyroid binding capacity by use of a fluorescent T.sub.3 tracer, such as T.sub.3 coupled to a fluorescein dye through a radical derived from L-lysine.

Abstract: The present invention relates to a method which eliminates centrifugation and decantation steps, to be performed in an automatic manner for carrying out specific binding assay tests, wherein liquid and solid phases are present.According to the invention, use is made of a specially designed device, consisting of a mixing reservoir into which is fitted snugly a mixer separator having a channel in the vertical axis of the mixer-separator. A rack holding a number of said mixing reservoirs containing the incubated reagents and analytes, capped with the mixer separators, is placed into a press-device designed to perform at a controlled rate a downward movement. The mixer separators are pushed downwards into the mixing reservoirs at a chosen rate for a preselected distance to complete the mass transport and separation operations. The separation devices are removed and either one of the separated phases can be measured in the desired analytical instrument for a quantitative or qualitative determination.

Abstract: There is described an enzyme immunoassay for measuring the concentration of an analyte in a sample wherein the substrate for the enzyme forms at least a part of the sample. In a particular embodiment the sample comprises or consists of a milk sample and the enzyme is an enzyme capable of clotting milk. An example given of such an enzyme is chymosin. The assay described may be used to measure the concentration of progestagens or oestrogens in milk using the techniques of heterogeneous or homogeneous enzyme immunoassay. The results of such an assay give an indication of the fertility of a milk producing domestic animal (e.g. a cow) and may be used to diagnose pregnancy of such an animal. Particular compounds for use in the assay are described, as is a kit of reagents for use in the assay.

Abstract: The vitamin D metabolite, calcitriol, is determined in blood serum by radioimmunoassay employing monoclonal antibodies in which calcitroic acid serves as the immunogen.

Abstract: A peptide having the formula ##STR1## is synthesized. The peptide is conjugated, e.g., with bis-diazotized benzidine, at its C-terminus to a carrier, such as bovine serum albumin, to form a synthetic antigen useful for inducing antibody production in a host animal. The antiserum obtained from the host animal is free of cross-reactivity with other pituitary substances and thus particularly advantageous for assay purposes. The peptide, whether unlabeled or labeled with radioactive iodine on the tyrosine moiety, has an affinity to antiserum raised against the conjugate similar to the affinity of natural hPRL to the antiserum. The synthetic peptide is used in radioimmunoassays where the labeled peptide competes for the binding sites in the antiserum with unknown concentrations of hPRL in biological samples.

Abstract: A test kit and assay for detecting the presence of minute amounts of an organic reactant in a test sample includes a plurality of beads or other types of support structures that are impregnated with a fluorescer and coated with a ligand that specifically binds to the organic reactant being investigated. The beads are placed in an aqueous solution, together with the reactant which has been radiolabeled. The portion of the radiolabeled reactant that binds to the ligand is thereby brought in close enough proximity to the beads to activate the fluorescer to produce light energy. The radiolabeled reactant that does not bind to the ligand is, for the most part, disposed too far away from the beads to enable the radioactive energy emitted thereby to reach the fluorescer integrated into the beads. Thus, the level of light energy produced by the fluorescer is indicative of the amount of reactant present in the test sample.

Abstract: A method to obtain a substantially pure and stable hLH-hCG receptor from gonadal cells is disclosed. The characteristics of the purified receptor are disclosed. The receptor, in addition to being used as receptor in an enzyme- or dye-receptor assay for hLH and/or hCG, can be used to purify hLH or hCG used in the preparation of standard dye-hormone or enzyme-hormone complexes. These complexes can be used in enzyme- or dye-immunoassays, as well as the receptor assay.

Abstract: Disclosed is a new mouse-mouse hybridoma tumor cell line A.T.C.C. No. HB8209. A monoclonal antibody produced by said cell line is specifically immunologically reactive with erythropoietin and with a polypeptide whose amino acid sequence is substantially duplicative of a sequence extant is erythropoietin. Disclosed also are procedures for isolation of erythropoietin by affinity purification and for quantitative detection of erythropoietin in fluid samples.

Abstract: In a particle agglutination assay for an antigen (Ag), there is included in the mixture a limited amount of a substance which binds univalently with a proportion of the Ag present, that Ag which is so bound being unable then to cause agglutination of the particles. In this way, unusually large concentrations of Ag can be assayed in that a proportion of the Ag is bound to the univalent substance and the particle agglutination assay is in effect conducted on the smaller amount of Ag still remaining free in solution.

Abstract: Immunological preparations are prepared by immunizing hens with an immunogen having a molecular or particle weight of at least 30,000, to a stage of hyper-immunization at which there occurs a plateau-like levelling-off of the antibody content of the serum. The immunogenicity of the immunogen can be enhanced by enlarging the immunogen particle mass. The eggs of the immunized hens are collected, the yolk is separated from the eggs, followed by separation of the lipid content of the yolk. The antibodies in the egg yolk are then rendered indispersable with the aid of a water-soluble linear filamentary non-charged polymer precipitant such as PEG and the indispersable antibodies are recovered. This precipitation of antibodies is advantageously preceded by a precipitation of caseinaceous proteins, lipid and yolk particles at lower polymer concentrations. Selected antibodies (IgY or IgG) can be concentrated by pH-controlled fractional precipitation with PEG.

Abstract: Competitive immunofluorescence assays for antigens in which immune complexes are precipitated with a nonfluorescent, nonlight-scattering precipitant such as polyethylene glycol and the resulting immunoprecipitate is dissolved with a nonfluorescent solvent of low ionic strength that maintains the pH of the solution substantially constant for immunofluorescence intensity reading. The assays are carried out by incubating the sample with fluorescent-labeled antigen, anti-antigen antibody, and a secondary antibody to the anti-antigen antibody followed by addition of the precipitant to form an immunoprecipitate. The precipitate is separated by centrifuging, dissolved in the solvent, and the immunofluorescence intensity of the solution is read with a fluorometer and compared to a standard curve.

Abstract: Ligand having at least two determinant or binding sites is contacted with a labeled first binder, an unlabeled second binder different than the first binder and a precipitating binder specific for the second binder, with such contacting precipitating a complex of the ligand and the first and second binders to thereby separate bound labeled binder from unbound labeled binder. The bound and/or unbound labeled binder is determined as a measure of the ligand.

Abstract: A highly sensitive and rapid method for quantitatively assaying analytes in liquid media by directly measuring changes in particle size distribution of reagent particles having analyte insolubilized thereon in a system undergoing antibody-induced aggregation has been developed. The amount of analyte initially present can be determined by measuring the change in the distribution of particle size with time, the concentration of a particular size particle at a given time, the rate of formation of a particular size particle, or the steady-state maximum concentration for a particular size particle.

Abstract: A novel analyte-cytolysin conjugate and its use in a lipid vesicle mediated measurement process is described for a wide variety of analytes present at very low concentration. The method involves forming a reaction system consisting of analyte, analyte specific binding agent, analyte-cytolysin conjugate, and vesicles containing detectable marker material in such proportions that uncombined conjugate alters the permeability of the vesicles resulting in the release and quantitative detection of marker material which can be correlated with the amount of analyte initially present.

Abstract: Mixtures of monoclonal antibodies which contain effective assaying amounts of each of at least two monoclonal antibodies that bind to different antigenic sites on the antigen and are capable under appropriate conditions of binding simultaneously to an antigen are useful in enhanced sensitivity assays for the antigen. By utilizing such mixtures in diagnostic assays for important antigens such as the polypeptide human chorionic gonadotropin enhanced sensitivity can be achieved as compared with assays employing individual monoclonal antibodies.

Abstract: This disclosure relates to a method for determining ligands in biological fluids such as serum, plasma, spinal fluid, amnionic fluid and urine, wherein a novel class of aminomethylfluorescein derivative tracer compounds are employed as reagents in fluorescence polarization immunoassays.

Abstract: There is described an assay for determining the relative concentrations of two antigenic solutes in a sample. The assay involves a first antibody to the first antigenic solute, a second antibody to the second antigenic solute and a ligand molecule having substituents to which the first and second antibodies may bind. The second antibody is immobilized upon a solid phase support and the other components are in solution. The components are mixed with a sample containing the antigenic solutes causing competition between the ligand molecule and each of the antigenic solutes for binding to the first and second antibodies. In the situation where there is a relatively higher concentration of the first antigenic solute to the second antigenic solute in the sample, a significant number of the ligand molecules become attached to the solid phase via the second antibody yet are capable of binding to a labelled antibody having the same specificity as the first antibody.

Abstract: A method is disclosed for extraction, concentration, and bioassay measurement of the concentration of bioactive parathyroid peptides in biological or other fluids. Parathyroid peptides are extracted from the fluid by a support-antibody matrix specific to the N-terminal region. The bioassay is directed to the extracted parathyroid peptides. The volume of the medium containing the extracted parathyroid peptides and tissue, cells or tissue extracts with adenylate cyclase-coupled parathyroid hormone receptors may be chosen to be significantly less than the volume of biological or other fluid being assayed.

Abstract: Colorimetric detection of bindable substances such as antibodies and antigens using chromogens of improved sensitivity and stability. The chromogens take the form of activated solutions containing tetramethylbenzidine or water soluble derivatives of tetramethylbenzidine. Such chromogens are of particular use in home testing applications for detection of antigens such as human chorionic gonadotropin, human luteinizing hormone, and gonococcus bacteria, as well as the detection of antibodies for these substances.

Abstract: An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques.

Abstract: A chromogen, in particular, a fluorescent compound, is conjugated to a ligand through an amino acid spacer radical, which includes a free carboxyl group. The tracers are particularly suitable for use in a flow through assay for low molecular weight analytes. The tracers are rapidly bound, of good sensitivity, and low non-specific binding.

Abstract: An immunoassay process for the detection of an antigen in a sample, which comprises: (a) forming a mixture of the sample with (1) a preformed complex of a primary antibody and a secondary binding macromolecule therefor, wherein the primary antibody is present at low concentrations and has substantial specificity for the antigen, the secondary binding macromolecule has substantial affinity for the Fc portion of the primary antibody, and the second binding macromolecule is affinity purified; and with (2) a detectably labeled form of the antigen; (b) incubating the mixture formed in step (a) for a time sufficient to allow the antigen and the detectably labeled antigen to competitively bind to the primary antibody of the preformed complex; (c) detecting the separated complex or the separated suspension medium.

Abstract: A method of forming an analyte-functionalized liposome for use in immunoassay techniques employs a phosphotriester intermediate of a phospholipid derivatized with the desired analyte in a process of forming a liposome which is functionalized on its outer surface with the analyte and which carries an enzyme marker. The method is also used to functionalize the liposome with a ligand which acts as a leash between the analyte and the liposome. Liposomes produced by the method include penicillin-G-functionalized liposome.

Abstract: A method for direct serum assay of steroid hormones wherein the competition from binding protein, having an affinity for complexing with the steroid hormones, is significantly eliminated by adding to the serum an artificial compound which has a greater or equal affinity for the binding protein than do the steroid hormones, and which is not immuno-reactive with the steroid hormones. Because the compound is artificial, it is non-contaminating. Levonorgestrel and norethindrone as such compounds are disclosed.

Abstract: A method of determining the concentration of an antigen in a sample solution comprising(a) coating an antigen-protein conjugate onto a solid matrix,(b) conjugating an enzyme to an antibody specific for said antigen,(c) to a known quantity of a solution containing the antibody-enzyme conjugate of (b) adding a specified quantity of a sample containing an unknown amount of the antigen whose content is to be determined,(d) contacting the coated solid matrix of (a) with the solution (c) and incubate so as to effect binding between the antibody and antigen, some of the antigen being that from the sample and some being that on the solid matrix,(e) removing the solid matrix from the solution and washing,(f) immersing the solid matrix in a solution containing a known amount of an enzyme-substrate which is acted upon by the enzyme so as to effect reaction between the enzyme and enzyme-substrate to produce a product, and then separating the solid matrix from the solution of enzyme-substrate, and(g) then measuring the so

Abstract: This invention relates to a highly-sensitive mini-iodinated radioactive hormone tracer which has both initial low iodine-to-hormone molar ratios and low specific activity. This invention also relates to methods for preparing and using such tracers in a radio-immunoassay which takes much less time than used heretofore and to a method of preparing said tracers.

Abstract: A method and kit for determining the presence of a polyvalent ligand in an aqueous fluid. The method involves incubating the fluid with an immobilized antibody to the ligand to form an immobilized ligand-antibody complex, then incubating the immobilized ligand-antibody complex with a solution of a soluble complex of a second antibody (to the ligand) and a labeled binding protein, such as protein A. The labeled binding protein is specifically bound to the Fc portion of the second antibody. The unbound second antibody and labeled binding protein are washed from the immobilized complex, and the presence of bound labeled binding protein is determined as a measure of the concentration of the ligand in the aqueous fluid.

Abstract: Immunogen conjugates comprising N-substituted-amino-3-desoxydigoxigenin derivatives coupled to conventional immunogenic carrier materials, and antibodies raised against such conjugates. Also provided are labeled digoxigenin conjugates for use with the digoxigenin antibodies in preferred immunoassay techniques for determining digoxin in biological fluids.

Abstract: The present invention relates to a method which eliminates centrifugation and decantation steps, to be performed in an automatic manner for carrying out specific binding assay tests, wherein liquid and solid phases are present.According to the invention, use is made of a specially designed device, consisting of a mixing reservoir into which is fitted snugly a mixer separator having a channel in the vertical axis of the mixer-separator. A rack holding a number of said mixing reservoirs containing the incubated reagents and analytes, capped with the mixer separators, is placed into a press-device designed to perform at a controlled rate a downward movement. The mixer separators are pushed downwards into the mixing reservoirs at a chosen rate for a preselected distance to complete the mass transport and separation operations. The separation devices are removed and either one of the separated phases can be measured in the desired analytical instrument for a quantitative or qualitative determination.

Abstract: The present invention relates to a new technique and device for mass transfer of one or more components from one liquid phase to another liquid phase, involving a physical separation of said two phases.According to the new technique, the mass transfer and physical separation are carried out in the same device. The device consists of a mixing-reservoir into which is fitted snugly a mixer-separator, having a channel in the vertical axis of the mixer-separator. The two substantially immiscible liquid solutions are introduced into the mixing reservoir, the phases are thoroughly mixed, by moving the mixer-separator in and out the mixing reservoir. After the spontaneous separation into an upper and lower phase, the upper phase is removed by pushing in the mixer-separator said upper phase being accumulated in a collecting container.

Abstract: This invention relates to a highly accurate, rapid and simple estimation of thyroxine (T.sub.4) directly from blood serum and also relates to the accurate measurement of triiodo-L-thyronine (T.sub.3) directly from blood serum. More specifically, the invention relates to a rapid, specific and reliable radioimmunoassay (RIA) technique for measurement of both T.sub.4 and T.sub.3 in unextracted serum. The method requires very small amounts of serum, e.g., 25 microliters (.mu.l) to measure T.sub.4 concentration in nearly all specimens representing clinical states of eu-, hypo- and hyperthyroidism, and 250 .mu.l to measure T.sub.3 concentrations in specimens representing most clinical states.