Studies on immunological assay of vitamin-K dependent factors.

Two-site immunoradiometric assays (IRMAs) for factor IX antigen (IX:Ag) were developed using a monoclonal antibody (RFF-IX/1) on the solid-phase and either another monoclonal antibody (RFF-IX/4) or a human polyclonal inhibitor antiserum as tracer (M-M and M-I IRMA respectively). The lower sensitivity limits of these two assays for IX:Ag in normal reference plasma were 4 X 10(-4) (M-M IRMA) and 2 X 10(-4) (M-I IRMA) units/ml. In 20 samples of normal plasma, levels of factor IX coagulation activity (IX:C) and of factor IX antigen measured by both IRMAs were highly correlated. Mean values of approximately 1.0 units/ml were obtained in all three assays. In normal serum, IX:Ag levels were lower with means of 0.84 (M-M IRMA) and 0.83 (M-I IRMA) units/ml. 4/25 patients with haemophilia B were CRM neg., two were CRM + and the remaining 19 patients were CRMr variants. In two of these, IX:Ag was detectable by M-I IRMA whilst IX:C and IX:Ag measured by M-M IRMA were undetectable. In plasma from a fetus subsequently terminated on eugenic grounds, IX:C and IX:Ag by both M-M and M-I IRMA were undetectable. In warfarin-treated plasma (n = 12), the level of IX:C was low (mean 0.39 units/ml). The levels of IX:Ag measured by M-M IRMA (mean of 0.80 units/ml) and by M-I IRMA (0.70 units/ml) showed a discrepancy. M-M IRMA reflects the real amount of IX:Ag in warfarinized plasma because both monoclonal antibodies bind to epitopes distant from the light chain carboxylated region. Western blotting of denatured factor IX demonstrated that RFF-IX/1 binds an epitope that is lost after XIa activation. RFF-IX/4 binds the heavy chain. Antigen measured after activation but without denaturing showed loss of 60% reactivity after XIa activation but no change after RVV activation. These data indicate a binding site for RFF-IX/1 within the activation peptide (residues 146-180).     

Two allotypes of factor IX present in haemophilia B

Factor IX antigen (IX:Ag) was measured with three different immunoradiometric assays (IRMAs) in 30 healthy people and 43 patients with haemophilia B of varying severity. Two of the IRMAs were based on monoclonal antibodies capable of differentiating between two genetically determined molecular variants of normal factor IX. Most patients with severe hemophilia B lacked demonstrable IX:Ag. The factor IX variant that is undetectable with one of the monoclonal antibodies used was present in 2 out of 6 families with moderate haemophilia B and in 1 out of 6 families with mild haemophilia B. The existence of allotypes of factor IX in hemophilia B may have practical implications for carrier detection and prenatal diagnosis.     

Detection of factor IX inhibitors by immunoradiometric assay

An immunoradiometric assay (IRMA) for the determination of factor IX inhibitors in haemophilia B was developed. The assay was based on competitive binding between radiolabelled anti-IX and inhibitors in the test material of immobilized IX:C. 5 haemophiliacs with known inhibitors were investigated with the new method and with a conventional neutralization test. An inhibitor titre as low as 5 X 10(-4) U/ml could be measured by IRMA, showing it to be about 500 times as sensitive as the conventional neutralization assay used, and in 2 cases the inhibitors could only be detected by IRMA. The new method is thus useful for the investigation of patients with low inhibitor titres, or when inhibitors with divergent properties are suspected.     

Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.     

Reevaluation of serum TSH determination in various pituitary disorders using highly sensitive immunoradiometric assay

In thirty-one patients with various pituitary disorders, their serum TSH levels were reevaluated using two kinds of highly sensitive immunoradiometric assays (IRMAs). TSH levels were measured by radioimmunoassay (RIA) supplied by Daiichi Radioisotope Laboratory and the IRMAs, which were RIA-gnost TSH Ultrasensitive and SUCROSEP TSH IRMA using two types of monoclonal antibodies. The sensitivities of them were 0.08 and 0.1 microU/ml, respectively. Free thyroxine and free triiodothyronine levels were measured by RIAs (Amerlex kits). There was a significant correlation between basal TSH levels and the maximum increase in serum TSH above baseline using the RIA and the IRMAs when responses to TRH (500 micrograms i.v.) was determined, but no correlation between basal TSH and thyroid free hormone levels. In their TRH tests, three patients showed absent responses to TRH which were measured by the RIA, although by the IRMAs slight responses were observed. In two patients, exaggerated responses to TRH were observed and the increase of TSH levels measured by the IRMAs showed 143 approximately 210% higher than that of the RIA. The IRMAs were able to handle their peak levels time in all the patients with responses, however, in some patients the RIA was not sensitive enough to distinguish it. These results indicated that measurement of TSH levels by the IRMAs might be useful in the evaluation of the TSH secretion capacity in patients with various pituitary disorders.     

Monoclonal antibody to an epitope on the heavy chain of factor IX missing in three hemophilia-B patients

A murine hybridoma cell line that produces a monoclonal IgG1 antibody to human factor IX was established to provide a conformational probe for the clotting factor and its genetic variants. The antibody inhibited factor IX procoagulant activity, but did not appreciably interfere with the cleavage of factor IX by factor XIa nor with the binding of antithrombin-III-heparin complex to factor IXa. The antigen- solid-phase-antibody complex could be readily dissociated by relatively low concentrations of guanidine or sodium dodecyl sulfate, but only partially by high concentrations of urea. After gel electrophoresis and blotting of reduced samples of factor IXa, the antibody bound exclusively to the heavy chain. Sensitive immunoradiometric assays were developed using insolubilized monoclonal or polyclonal antibodies. Bovine factor IX had little cross-reactivity with the monoclonal antibody. Of 55 patient samples representing different pedigrees with hemophilia-B, antigen levels by the two assays were in excellent agreement in 49. There were 2 severely affected patients whose levels were too low to quantitate in the monoclonal antibody assay. A third, who had the lowest level of all by polyclonal antibody testing, and 3 less severely affected patients had no detectable antigen in the monoclonal antibody assay system (less than 0.03 U/dl). The latter 3 had at least 100-500 times as much antigen by polyclonal antibody testing. It is proposed that these 3 individuals have structural defects involving the epitope recognized by the monoclonal antibody and that they are due to amino acid substitutions between residues 188 through 359. Furthermore, it is suggested the substitutions lead to abnormal kinetic properties.     

Immunoassays of factor IX antigen using monoclonal antibodies

Monoclonal antibodies to factor IX were produced by immunization of balb/c mice with purified factor IX and fusion of spleen cells with SP-1 murine myeloma cells. Antibody producing hybrids were detected by an enzyme linked immunoassay (ELISA) and by coagulation inhibitor (Bethesda type) methods. Monoclonal antibodies with titres of greater than 1 X 10(5) tested by the ELISA and 5000-8000 inhibitor units were obtained. A new ELISA method was developed using one of these monoclonal antibodies to quantitate factor IX antigen (IXAg) in plasma samples from patients with hereditary factor IX deficiency. In addition a two-site solid phase immunoradiometric assay (IRMA) for factor IX was established. The results of these new methods were compared with those obtained on the same plasma samples using conventional factor IX coagulation assays and the Laurell rocket method. The lower limit for the detection of IXAg by the ELISA was approximately 0.01 unit per ml whilst that for the IRMA was about 0.001 unit per ml (normal plasma = 1 unit per ml). The lower detection limit for IXAg using the Laurell rocket method was about 0.06 units per ml. The improved sensitivity of the new immunoassays enabled quantitation of low levels of IXAg in patients with moderately severe factor IX deficiency and confirmed the presence of excess IXAg compared to IX activity (IXC) in a relatively high proportion of cases (28 out of 51 tested). Results of testing plasma from obligate carriers confirm the suggestion that measurements of IXAg and IXC may improve the classification of carrier status in these kindred.     

A new generation IRMA for ACTH with improved specificity: validation in various physiological and pathological conditions

OBJECTIVE: Measurement of plasma ACTH is a key step for the exploration of hypothalamic-pituitary-adrenal disorders. To further improve ACTH recognition a new generation of ACTH IRMA was developed using antibodies directed towards succinylated ACTH (sACTH IRMA). DESIGN: The usefulness of this assay was compared with that of another commercially available ACTH IRMA assay using intact ACTH (ELSA-ACTH) in various pathophysiological situations: patients with low ACTH plasma levels, high ACTH plasma levels with normal or tumoural pituitaries, or ectopic ACTH syndrome, and pregnant women with high proopiomelanocortin (POMC) plasma levels. METHODS: All plasma samples were assayed simultaneously with the two different IRMAs. Comparisons were assessed by plotting the results along the theoretical line of identical values, and by the graphical method of Bland and Altman. RESULTS: In the ELSA-ACTH IRMA, CLIP (or ACTH18-39) showed true cross-reactivity, and alpha-melanocyte-stimulating hormone and purified POMC both interfered and induced falsely lower ACTH results; in the sACTH IRMA no peptide showed any cross-reactivity, and only extremely high values of CLIP (50 000 pg/ml) interfered and induced falsely lower ACTH results. In ACTH hypersecretory syndromes, of tumoural (Cushing's disease, ectopic ACTH secretion) or non-tumoural (Addison's disease, congenital adrenal hyperplasia) origins a good agreement between the two assays was observed except for very high ACTH plasma values (above 1000 pg/ml) and in some tumours where the sACTH IRMA yielded lower results; in some cases, the presence of circulating CLIP, demonstrated by HPLC studies, may contribute to this discrepancy. It is also likely that the calibration of the ELSA-ACTH kit itself generates higher ACTH values. In normal pregnant women both IRMAs gave highly correlated values, yet lower results were obtained with the sACTH IRMA. CONCLUSION: These data show that the sACTH IRMA has improved qualities of specificity and usefulness for rapid assessment of ACTH plasma levels.     

Defective propeptide processing and abnormal activation underlie the molecular pathology of factor IX Troed-y-Rhiw

Affected members of a South Wales haemophilia B family (from Troed-y-Rhiw) were shown by Western blotting and immunoperoxidase detection to have a factor IX molecule of higher than normal molecular weight which also shows impaired calcium binding. Gene cloning and DNA sequencing revealed the same arginine to glutamine mutation at position -4 of the propeptide that has been found in two previously described factor IX variants which circulate with the propeptide still attached. The mutation also abolishes a HaeIII restriction enzyme recognition site. A potential carrier was shown to be normal both by Western blotting and DNA studies. The way in which the attached propeptide interferes with normal factor IX function was investigated by activation studies with crude normal and patient factor IX Troed-y-Rhiw preparations using Western blotting and detection with iodinated immunopurified polyclonal antifactor IX serum. We demonstrate that the -4 mutation appears to block cleavage between the Arg145-Ala146 peptide bond in the activation peptide, thus preventing the normal activation of factor IX Troed-y-Rhiw. A small amount of normally processed factor IX is produced, implying that the -4 mutation does not completely prevent propeptide cleavage, thus accounting for the low levels of factor IX activity measured in the plasma of affected family members.     

Variability in the quantitation of circulating growth hormone using commercial immunoassays

The quantitation of human GH in a serum sample is not consistent among various commercially available immunoassays. We measured serum GH concentrations with four RIAs [Cambridge, Kallestad, National Hormone and Pituitary Program, and Radioassay Systems Laboratories (RSL)] and two immunoradiometric assays (IRMAs; Hybritech and Nichols). Serum GH concentrations measured by the RIAs were between 1.9 and 2.8 times higher than those determined by the Hybritech IRMA, whereas the concentrations measured by the Nichols IRMA were approximately 3.0 times higher than the Hybritech values. We evaluated the effects of differences in standards, assay diluents, and antibody specificity on GH measurement in the various assays. When GH standards from each of the assays were measured in the Hybritech IRMA, only the RSL standard was less immunoreactive than the other assay standards. Different assay diluents also resulted in varying GH values. In the RIAs, GH diluted in serum was more immunoreactive than GH diluted in phosphate-buffered saline-0.5% BSA. This enhanced immunoreactivity appeared to be due to a nonspecific effect generated by serum. The Nichols and Hybritech IRMAs provide standards diluted in horse serum. In the Nichols assay, GH diluted in human serum was more immunoreactive than GH diluted in horse serum, whereas the immunoreactivity of GH diluted in either serum was equal in the Hybritech IRMA. These IRMAs also differ in that the Nichols assay detected the 20K variant of GH, whereas the Hybritech assay did not. Considering these discrepancies, comparison of data obtained using different assays should be made carefully.     

Antigenic determinant in human coagulation factor IX: immunological screening and DNA sequence analysis of recombinant phage map a monoclonal antibody to residues 111 through 132 of the zymogen

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX- 30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.     

Determination of factor IX allotypes for carrier identification in haemophilia B

The existence of two genetic variants (allotypes) of normal human factor IX is used for carrier detection in three families with severe and one with mild haemophilia B. By analysis of IX:Ag with two different monoclonal antibodies in 93 members of the families, allelic assignment is shown to be a complement in carrier diagnosis to genotypic DNA studies. Allelic assignment makes possible a reliable diagnosis based on phenotypic studies, though its usefulness is limited due to ethnic variation in allelic frequency. Determination of factor IX allotypes should be useful for carrier detection in many Swedish families with haemophilia B.     

Potent cross-group neutralization of primary human immunodeficiency virus isolates with monoclonal antibodies--implications for acquired immunodeficiency syndrome vaccine

Human immunodeficiency virus type 1 (HIV-1) is phylogenetically classified into groups and clades (or subtypes). Human neutralizing monoclonal antibodies (nMAbs), originally isolated from individuals infected with HIV-1 group M-clade B, neutralized not only primary HIV-1 clade B isolates in vitro but also primary isolates of other group M clades (A, C, D, E, and F). This corrected the previously held notion that primary HIV-1 isolates are resistant to neutralizing antibodies. Here we show that anti-HIV-1 group M-clade B nMAbs potently neutralized primary isolates of the phylogenetically distant HIV-1 group O. We and others have previously shown that passive immunization with human nMAbs protected adult or neonatal primates against infection with simian-human immunodeficiency virus strains encoding HIV-1 group M-clade B envelope genes. The in vitro cross-group neutralization shown here underscores the broad potential of these nMAbs against divergent virus variants and the relevance of their epitopes in the design of acquired immunodeficiency syndrome vaccines.     

Clinical application of a sensitive immunoradiometric assay for human growth hormone

A sensitive immunoradiometric assay (IRMA) for human growth hormone (GH) using monoclonal antibodies was evaluated for clinical application. The detection limit of GH in serum was 0.05 ng/ml using 0.1 ml of serum. The specificity and assay precision were satisfactory. GH levels in serum determined by the IRMA were well correlated to those determined by the sensitive enzyme immunoassay or the commercial radioimmunoassay. Most of the basal GH levels in serum from normal adult subjects were detectable; mean values in male and female were 0.21 and 0.65 ng/ml, respectively. Serum GH levels decreased to less than 1 (0.5) ng/ml after oral glucose loading in normal subjects. Postoperative acromegalic patients were classified into two groups according to their GH responses to glucose. In group 1, GH levels were suppressed less than 1 ng/ml, suggesting normal GH dynamics. On the other hand, in group 2, GH levels remained elevated above 1 ng/ml, and postinhibitory rebound rise in serum GH after dopamine infusion was observed, suggesting the presence of residual adenoma tissues. Determination by sensitive IRMA of GH responses during oral glucose loading is useful for evaluating the activity of postoperative acromegaly.     

Spectrum of HTLV-III infection in a hemophilic cohort treated with blood products from a single manufacturer

One hundred fifty-eight hemophilia A, B, and von Willebrand disease (VWD) patients treated with clotting factor concentrates from a single manufacturer were tested for antibody to the human T-lymphotropic virus type III (HTLV-III). Antibody was detected in 63% and 40% of those with severe hemophilia A and B, respectively, 12% and 0% of those with mild hemophilia A and B, and two patients with recessive VWD. Forty-two antibody-positive and 20 antibody-negative patients were studied for clinical and laboratory features of infection. Eleven seropositive patients had clinical signs of infection including Pneumocystis carinii pneumonia, lymphadenopathy, splenomegaly or diarrhea; however, only one patient had developed acquired immune deficiency syndrome (AIDS), and only two had significant impairment of their performance status. Thirty-one patients remained totally asymptomatic. Eight patients had a history suggestive of acute HTLV-III infection. Thrombocytopenia was observed in 18% of seropositive patients, lymphopenia in 60%, depressed T-helper cells in 43%, reduced T-helper:T-suppressor ratios (TH:TS) in 33%, and elevated platelet-bound immunoglobulin in 53%. The antibody-negative group had normal T-helper cell levels (except one patient) and TH:TS ratios, and normal platelet immunoglobulin levels. Both groups demonstrated a significant elevation of immunoglobulin levels and a high prevalence of antinuclear factor and antismooth muscle antibodies. The mean level of IgG was significantly higher in the antibody-positive group. This study confirms the correlation between HTLV-III infection and reduced T-helper cells in hemophiliacs but demonstrates a low incidence of clinical symptomatology. There was evidence of polyclonal B-cell hyperactivity in the antibody-negative group as well as the seropositive group.     

Gonadotropin evaluation in the diagnosis of polycystic ovary syndrome using either a monoclonal or a polyclonal antibody radioimmunoassay.

The LH/FSH ratio values between gonadotropins dosed with a monoclonal antibody assay (IRMA) in the micropolycystic ovary syndrome (PCOS), are discussed and compared to those obtained with the classic assays using polyclonal antibodies. Because of the higher selectivity of this IRMA assay it is noteworthy that the cut-off value between normal and PCOS patients is now equal to or above one. The evaluation of the LH/FSH ratio between the peak values of the two gonadotropins after a GnRH 100 micrograms iv bolus, may be useful in the diagnosis of PCOS in those patients who present an LH/FSH less than 1 in basal conditions even in the presence of clinical and ecographic aspects of PCOS.     

The combined use of monoclonal antibody- based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haernophilic plasmas tested, VIII: Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII: C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII: Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF: Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII: C/vWF: Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII: Ag and vWF: Ag.     

Immunochemical characterization of a polyclonal human antibody to factor IX.

Inhibitors of clotting factors occuring in humans are often antibody molecules synthesized in response to exogeneous proteins used in replacement therapy. Extensive studies of inhibitors to factor VIII indicate such antibodies may be monoclonal or polyclonal in nature. To date, only one factor IX inhibitor has been subjected to detailed immunochemical analysis and it appears to be a monoclonal IgGA lambda antibody. We have discovered a second inhibitor of factor IX in a patient with severe hemophilia B and have subjected it to immunochemical analysis. Studies on this second inhibitor have been carried out before and after an anamnestic response. Column chromatography, preparative zone electrophoresis, and specific inhibitor neutralization assays using monospecific heterologous antisera to human immunoglobulin classes, subclasses, and light-chain types indicate that the antibody is of the IgG class and contains both kappa and lambda light chains and probably all four IgG subclasses. Thus, the inhibitor appears to be polyclonal by immunochemical and structural criteria. In addition, preparative isoelectric focusing of pre- and postanamnestic inhibitor samples indicates that recruitment of new clones of IgG antibody occurs as a result of anamnesis. It is conceivable that an antibody initially restricted in immunoglobulin subclass became polyclonal following an anamnestic response.     

Hyperinsulinaemia in the polycystic ovary syndrome confirmed with a specific immunoradiometric assay for insulin

OBJECTIVE Hyperinsulinaemia in the polycystic ovary syndrome (PCOS) has previously been defined using polyclonal radioimmunoassays (RIA) in which partially processed insulin-like molecules cross-react. This study aimed to reassess hyperinsulinaemia in women with PCOS using specific immunoradiometric assays (IRMA) for insulin, proinsulin and 32–33 spilt proinsulin. DESIGN Patients attended for 75 g oral glucose tolerance tests and were divided into groups depending on their degree of obesity and fasting insulin status determined by RIA. IRMA measurements for insulin-like molecules in plasma from patients with PCOS and controls were compared. PATIENTS Thirty-four patients with ultrasound diagnosed PCOS presented to a reproductive endocrinology clinic. A control group comprised women with normal ovaries on ultrasound. Four groups were constructed, two with normal fasting insulin concentrations (lean PCOS and controls) and two with hyperinsulinaemia (lean and obese PCOS). MEASUREMENTS Plasma glucose, insulin (RIA and IRMA), proinsulin and 32–33 split proinsulin concentrations were measured at time 0, 30 and 120 minutes of an oral glucose tolerance test. RESULTS Hyperinsulinaemia determined by RIA in lean and obese women with PCOS was confirmed using a specific IRMA assay for insulin. Plasma proinsulin and 32–33 split proinsulin concentrations were higher in hyper-insullnaemic women with PCOS compared with women with normal insulin concentrations. The proportion of circulating insulin-like molecules represented by proinsulin and 32–33 split proinsulin was similar in all groups studied. CONCLUSIONS Hyperinsulinaemia in PCOS is likely to reflect insulin resistance because the raised concentrations of proinsulin and 32–33 split proinsulin were in proportion to the raised insulin concentrations. Hyperinsulinaemia in PCOS, defined by RIA, therefore differs from that in non-Insulin dependent diabetes mellitus where it Is largely accounted for by disproportionate hyperproinsulinaemla.     

Alloantibodies to Factor IX in Haemophilia B Characterized by Crossed Immunoelectrophoresis and Enzyme-conjugated Antisera to Human Immunoglobulins

Incubation of factor IX with non-precipitating alloantibodies to factor IX gives rise to soluble complexes between factor IX and the alloantibodies. These complexes appear as a factor IX molecule with a reduced mobility in crossed immunoelectrophoresis against a rabbit antiserum to factor IX. This factor IX immunoprecipitate was used for the study of alloantibodies to factor IX from five patients with severe haemophilia B (antibody titres 0.1--800 units/ml plasma). Incorporation of antiserum to k light chains or lambda light chains of human immunoglobulin G in an intermediate gel in crossed immunoelectrophoresis gave a reduction of the factor IX precipitin arc, indicating the presence of immunoglobulin G alloantibodies containing both k and lambda light chains in complex with factor IX. Incubation of the factor IX immunoprecipitate with peroxidase-conjugated antisera to the same immunoglobulins, and staining for peroxidase activity, confirmed the presence of immunoglobulin G containing both types of light chains in the factor immunoprecipitate. It is concluded that all five alloantibodies were polyclonal immunoglobulin G antibodies. The technique had the advantage that the light chain types of low-titre antibodies could be determined, and it may be suitable for further characterization of alloantibodies to factor IX if antibodies to immunoglobulin subclasses are available.