Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation.

Changes in the lactoferrin and lysozyme concentration of human milk during lactation were determined by microparticle-enhanced nephelometric immunoassays of 360 milk samples collected from 64 lactating volunteers. These 360 samples were colostrum from days 1 to 5 postpartum (142 samples), transitional milk from days 6 to 14 (106 samples), and 112 mature milk samples obtained from days 15 to 28 (34 samples), from days 29 to 56 (50 samples) and from days 57 to 84 postpartum (28 samples). The concentration and percentage of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.8 g/L, 27%) than in transitional milk (3.1 g/L, 22%) or day 15 to 28 mature milk (2.0 g/L, 19%), then increased in day 29 to 56 mature milk (2.2 g/L, 22%) and day 57 to 84 mature milk (3.3 g/L, 30%). The concentration of lysozyme decreased from colostrum (0.37 g/L) to transitional milk (0.27g/L) and day 15 to 28 mature milk (0.24 g/L), then increased in day 29 to 56 mature milk (0.33 g/L) and was highest in day 57 to 84 mature milk (0.89 g/L). The percentage of lysozyme vs. total protein was found to be always rising during lactation: colostrum, 2%; transitional milk, 2%; days 15 to 28, 2%; days 29 to 56, 3%; and days 57 to 84 mature milk, 8%.

Sequential C3 and C4 levels in human milk in relation to prematurity and parity.

Similarly to many immune molecules of human milk, C3 and C4 levels decrease during lactation. We investigated the influence, over the first three weeks of lactation, of both prematurity and parity on the sequential evolution of these levels. Milk C3 and C4 concentrations were measured by immunonephelometry in 494 individual samples collected from 76 lactating mothers. C3 and C4 concentrations were higher in milk from preterm or primiparous mothers. The major differences were observed in milk from days 5-8 and 9-20, likely due to pronounced interindividual variations in levels of days 1-4 milk. Milk from mothers of precocious (33 weeks' gestation or less) preterm newborns presented higher concentrations and a slower decrease of C3 and C4 levels than that from mothers of late (33-37 weeks' gestation) preterm newborns, when compared to term mothers. Finally, the inversion of the C3/C4 ratio occurring over time, previously reported, appeared later in milk from mothers of preterm newborns. The influence of prematurity was even greater in primiparous than in multiparous mothers. Both C3 and C4 levels therefore appear to be influenced in human milk by the parity and prematurity of the delivery. Mothers from preterm newborns seem to provide higher levels of C3 for a longer period post delivery.

Measurement of nine human milk proteins by nephelometric immunoassays: application to the determination of mature milk protein profile.

OBJECTIVES: Microparticle-enhanced nephelometric immunoassays for six human milk proteins (beta-casein, kappa-casein, alpha-lactalbumin, serum albumin, lactoferrin, and lysozyme) and conventional immunonephelometry assays for immunoglobulin A, C3, and C4 complement proteins were developed and characterized. DESIGN AND METHODS: Microparticle-enhanced nephelometric immunoassays are competitive assays based on the nephelometric quantification of the inhibition of microparticle-protein conjugates immunoagglutination by the proteins to be assayed. RESULTS: High precision (CVs ranged from 1% to 14% in within- and between-assays) and recovery (linear recovery in dilution-overloading assay) ensure a reliable determination of the main human milk proteins by single-step homogeneous nephelometric immunoassays, accurate over wide ranges of concentration. These immunoassays were easily applied to a large number of mature human milk samples (between 373 and 503 according to the proteins tested). CONCLUSIONS: The immunoassays developed could be applied to the fast determination of human milk protein profile usable for nursery milk bank and fortification.

C3/C4 concentration ratio reverses between colostrum and mature milk in human lactation.

The levels of complement fractions C3 and C4 were assayed in human milk in a classic nephelometric assay adapted to this secretion. Concentrations of these molecules were measured in 667 milk samples obtained sequentially from 76 volunteer lactating mothers during the first 12 weeks of lactation. Immunonephelometry was performed using skimmed milk samples diluted 10 times and yielded reproducible (coefficients of variation in within- and between-run precision lower than 9% for C3 and than 14% for C4) and accurate (linear recovery in dilution-overloading assay) data. High concentrations (mean +/- SE) were found for C3 (199.32+/-16.35 mg/L) and C4 (113.42+/-11.16 mg/L) in colostrum samples (n = 159; days 1-5). A significant (P<0.001) and rapid decrease was observed in transitional milk samples (n = 198; days 6-14), containing 57.71+/-5.18 and 72.39+/-4.98 mg/L of C3 and C4, respectively. Stable lower levels were noted in mature milk samples (n = 310; days 15-84) at 30.36+/-1.57 mg/L for C3 (P<0.001) and 53.38+/-3.61 mg/L for C4 (P<0.05). The decrease rate was different for C3 and C4, yielding a reversal of the C3/C4 ratio between colostrum and more mature milk.

Changes in the kappa-casein and beta-casein concentrations in human milk during lactation.

A microparticle-enhanced nephelometric immunoassay was developed for kappa-casein quantification in human milk. Together with a previously reported beta-casein comparable immunoassay, it was applied to 862 samples milk, collected from 82 mothers, to investigate the changes in casein concentrations in human milk during the first twelve weeks of lactation. kappa-casein immunoassay is sensitive (detection limit in the reaction mixture, 0.02 mg/L) and can be performed in diluted milk, excluding any interference or sample pretreatment. It allowed the quantitation of kappa-casein over a large range of concentrations (0.14-4.56 g/L) with accuracy and precision (coefficients of variation from 3 to 10%). beta- and kappa-casein concentrations and percentages among milk total proteins increase between colostrum (2.6 g/L, 14.3% and 1.2 g/L, 6. 5%, respectively) and transitional milk (4.4 g/L, 33.2% and 1.3 g/L, 9.5%), decrease at different rates from the third to the eighth week, then remain stable at least up to the end of the third month of lactation (2.7 g/L, 25.3% and 0.9 g/L, 8.5%). The beta-casein/kappa-casein ratio is higher in colostrum (0.61) than in transitional and mature milk (0.30) and could be related to a better digestibility of colostrum casein micelles by the neonate during the first days of life.

Immunological and nutritional composition of human milk in relation to prematurity and mother's parity during the first 2 weeks of lactation.

BACKGROUND: To investigate the effect of prematurity and parity on the dynamics of the major immunologic and nutritional proteins of human milk over the first 2 weeks of lactation. METHODS: Microparticle-enhanced nephelometric immunoassays were developed for the quantification of alpha-lactalbumin, beta-casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A, and total proteins were assayed in 368 individual samples collected from 74 mothers. RESULTS: The dynamics of the major immunologic and nutritional proteins in early lactation presented similar patterns in preterm and term human milks. In comparison with term milk, preterm milk was globally characterized by higher concentrations of immune proteins and lower concentrations of nutritive proteins. These differences were increased by the degree of prematurity, which, however, influenced the absolute and relative protein concentrations differently, depending on the stage of lactation. The protein composition of term milk was similar, whatever the mother's parity. Conversely, the influence of prematurity on the levels of milk proteins during the first days of lactation was even greater in primiparous mothers. CONCLUSIONS: This precise description of the composition of preterm and term milk, regarding the main nutritional and immunologic proteins, confirms the influence of both prematurity and parity on milk components and demonstrates the combined effect of these two conditions.

[Hospital results of angioplasty in multiple coronary vessel disease in 1990-1991 and 1994-1995. What conclusions should be drawn about the improvement of results with respect to the relevance of data of randomized trials comparing angioplasty and surgery]. / Résultats hospitaliers de l'angioplastie chez les multitronculaires en 1990-1991 et en 1994-1995. Quelles conséquences tirer de l'amélioration des résultats quant à l'applicabilité des données des études randomisées comparant angioplastie et chirurgie?

The aim of this study was to determine whether advances in angioplasty techniques have improved results in multiple vessel coronary disease and to compare present results with those reported in randomised trials comparing angioplasty and surgery. The hospital results of two cohorts of multivessel coronary patients treated by angioplasty during two different periods were compared (group 1: 1990-1991. group 2: 1994-1995). The first period corresponded to the inclusion period of randomised trials comparing surgery and angioplasty. The patients in group 2 (n = 449) were older than those in group 1 (n = 424), had more triple vessel disease, more severe angina and more previous angioplasty attempts. Moreover, there were more cases of unfavourable lesions. Nevertheless, the clinical success rate was high in group 2 (92% vs 84%; p < 0.001) and the major complication rate (death, myocardial infarction or emergency bypass surgery) was lower (2.9% vs 6.1%; p = 0.02). The main technical difference between the two periods concerned the use of coronary stents (12% vs 8%; p < 0.001). The fact of being in group 2 was identified by multivariate analysis as an independent predictor for clinical success and a lower major complication rate. The authors conclude that, since the publication of randomised trials comparing angioplasty with coronary surgery, the hospital results of angioplasty have significantly improved. This should be taken into account in considering the clinical applications of the results of these trials.

Microparticle-enhanced nephelometric immunoassay of lysozyme in milk and other human body fluids.

Quantitation of lysozyme in human milk was performed by a microparticle-enhanced nephelometric immunoassay based on the measurement of the light scattered during the competitive immunoagglutination of a microparticle-lysozyme conjugate with an anti-lysozyme antiserum. This immunoassay has a detection limit of 8 microg/L of reaction mixture and can be performed using diluted milk (1:6000, in reaction mixture), excluding sample pretreatment. Human milk lysozyme can be quantified over the concentration range 0.09-1.50 g/L, with within- and between-run coefficients of variation <5%. Changes in the lysozyme concentration of human milk during lactation were determined in 636 samples. Lysozyme concentrations (mean +/- SE) decreased from colostrum (0.36 +/- 0.02 g/L) to transitional milk (0.30 +/- 0.01 g/L) and mature milk during days 15-42 (0.30 +/- 0.01 g/L), then increased in the mature milk during days 43-56 (0.35 +/- 0.01 g/L) and especially during days 57-84 (0.83 +/- 0.05 g/L). The proportion of lysozyme contributing to total protein was found to rise during lactation and was as follows: colostrum (1.7%), transitional milk (2.3%), and mature milk from days 15-28 (2.7%), days 29-42 (3.1%), days 43-56 (3.8%), and days 57-84 (7.3%). The assay developed for milk was also suitable for the determination of lysozyme in other human body fluids.

Changes in immediate outcome of percutaneous transluminal coronary angioplasty in multivessel coronary artery disease in 1990 to 1991 versus 1994 to 1995.

Coronary angioplasty has undergone major technical changes since the period of inclusion in the randomized trials, comparing it with surgery, particularly with the increased use of coronary stents. This study shows improved in-hospital outcome in terms of primary success and complication rates in patients treated with coronary angioplasty for multivessel disease from 1994 to 1995, compared with the 1990 to 1991 period.

Microparticle-enhanced nephelometric immunoassay of alpha-lactalbumin in human milk.

A microparticle-enhanced nephelometric immunoassay was developed for alpha-lactalbumin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-alpha-lactalbumin conjugate with an anti-alpha-lactalbumin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 1.5 micrograms/L) and could be performed in high dilution of milk, excluding any interference or sample pretreatment. It allowed the quantification of alpha-lactalbumin on a large range of concentrations (0.5-16.9 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 1 to 7%). Changes in the alpha-lactalbumin concentration of human milk during lactation were determined in 162 samples. The concentration and ratio of alpha-lactalbumin total protein were found to be significantly lower in colostrum (4.9 g/l, 27%) than in transitional milk (5.2 g/L, 40%), then decreased in mature milk (3.4 g/L, 31%).

Microparticle-enhanced nephelometric immunoassay of lactoferrin in human milk.

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immuno-agglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675-21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%).

Effect of late percutaneous angioplastic recanalization of total coronary artery occlusion on left ventricular remodeling, ejection fraction, and regional wall motion.

The clinical benefit of late recanalization of complete coronary occlusion is debated. Left ventricular (LV) function and volumes are major prognostic determinants in patients with coronary artery disease. We sought to assess comprehensively the evolution of global and regional LV function and LV volumes after percutaneous recanalization of chronic complete coronary artery occlusions. A consecutive series of 55 patients who underwent successful percutaneous recanalization of a chronic (> or = 10 days), total (Thrombolysis in Myocardial Infarction trial flow grade 0) occlusion of the left anterior descending or dominant right coronary arteries, and in whom a complete angiographic evaluation was available before angioplasty and at follow-up was studied. At follow-up, 38 patients had a patent artery (group 1) and 17 had a reocclusion (group 2). Baseline parameters were similar in the 2 groups. In group 1, LV ejection fraction increased from 55 +/- 14% to 62 +/- 13% (p <0.001), with an increase in fractional shortening in the occluded artery territory (0.43 +/- 0.30 to 0.71 +/- 0.34, p <0.001), while LV end-diastolic volume remained unchanged. In group 2, ejection fraction and regional wall motion were unchanged, while LV end-diastolic volume index increased (86 +/- 22 ml/m2 to 99 +/- 34 ml/m2, p <0.02). The evolution in LV global and regional function was similar in patients with or without previous myocardial infarction; however, prevention of LV remodeling was observed only in patients with previous infarction. Maintained potency after successful recanalization of totally occluded coronary arteries improves global and regional LV function and, in patients with previous myocardial infarction, avoids LV remodeling.

Microparticle-enhanced nephelometric immunoassay of human plasminogen.

A microparticle-enhanced nephelometric immunoassay was developed for plasminogen quantitation in human plasma. It is based on the nephelometric measurement of the light scattered by microparticle clusters formed during a sandwich reaction between plasminogen, microparticle--anti-plasminogen conjugate, and the free antibodies of anti-plasminogen rabbit antiserum. This immunoassay was sensitive (detection limit in reaction mixture, 34 micrograms/L) and could be performed in 500-fold diluted human plasma, excluding any interference or sample pretreatment. It allowed the quantitation of plasminogen on a large range of concentrations (17-550 mg/L), with a security in antigen excess reaching 1,100 mg/L, with accuracy (linear recovery in dilution-overloading assay and correlation with conventional immunonephelometry), and precision (within- and between-run coefficients of variation lower than 8%). A normal reference range from 54 to 148 mg/L (mean +/- 2 SD) was calculated from plasminogen concentration in plasma from 130 adults. Easy to perform (no washing or phase separation) and rapid (two steps of 30 minutes then 1 hour of incubation at room temperature), this microparticle-enhanced nephelometric immunoassay could be an interesting alternative method for human plasminogen quantitation.

Changes in patient treatment after abrupt closure complicating percutaneous transluminal coronary angioplasty: a historic perspective.

This study compares the incidence and management of acute closure complicating coronary angioplasty in three historic populations of patients having undergone the procedure at the same center: group 1 (n = 146 of 881) ("early years" of angioplasty, 1980 to 1986), group 2 (n = 113 of 1781) (bailout stenting learning curve, 1990 to 1992), and group 3 (n = 34 of 525) (1993). The incidence of acute closure decreased from group 1 (146 [17%] of 881) to groups 2 and 3 (147 [6%] of 2306); (p < 0.001). Management of the occlusion changed over the years, with less emergency coronary bypass surgery ([36%] 52 of 146, 15 [13%] 113, and 3 [9%] of 34), respectively, p < 0.01) and more repeat angioplasty (70 [48%] of 146; 87 [78%], of 113, and 30 [88%] of 34, p < 0.001). The use of prolonged inflations (> 10 minutes) and stenting increased from group 2 (15 [13%] of 113 and 16 [14%] of 113, respectively) to group 3 (12 [35%] of 34, and 10 [30%] of 34, respectively). In-hospital death occurred in 18 (12%) of 146, 7 (6%) of 113), and (2 (6%) of 34) patients in the three groups. Acute myocardial infarction decreased from 64% to 46% and 27%, respectively (p < 0.01). Overall, the number of patients free of events at hospital discharge increased from 38 (26%) of 146 to 53 (47%) of 113 (p < 0.001) and to 23 (68%) of 34 (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

[Is percutaneous transluminal coronary angioplasty in chronic total coronary occlusion justified? Long term results in a series of 201 patients]. / L'angioplastie transluminale percutanée des occlusions coronaires totales chroniques est-elle justifiée? Résultats à long terme d'une série de 201 patients.

Percutaneous transluminal coronary angioplasty of chronic total coronary occlusions has a low primary success rate and is associated with a high percentage of restenosis. The aim of this retrospective study was to assess the long-term benefits of these procedures. In a series of 201 patients with 203 chronic total occlusions, the technical success rate was 51%, the clinical success rate was 46% with 3% of major complications. The only factor associated with a favourable outcome was the presumed duration of the occlusion. The clinical follow-up period was established at 6 years. The result of the initial procedure was used to establish two groups of patients: group I, clinical success, and group II, clinical failure. Patients in group I had a probability of survival greater than that of those in group II (97 vs 92%; p < 0.05); survival without coronary bypass surgery was also significantly better (89 vs 74%; p < 0.003). On the other hand, the probability without angioplasty was less in group I (70 vs 77%; p < 0.01), the result of a high restenosis rate (48%). A Cox analysis identified clinical success of angioplasty as a good prognostic factor for survival. Moreover, the clinical status at long-term was significantly better in patients in group I. These results indicate that in patients with chronic total coronary occlusions, the success of angioplasty has a favourable effect on long-term outcome both in terms of survival and in quality of life. They must be interpreted in the light of the limitations inherent in a retrospective study and should be confirmed by prospective trials.

[Does recanalisation of chronic right coronary occlusion improve long-term quality of life and the return to work?]. / La désobstruction des occlusions chroniques coronaires droites améliore-t-elle la qualité de vie à long terme et la reprise du travail?

Between 1985 and 1990, right coronary artery recanalisation was attempted in 60 consecutive patients. In order to evaluate the long-term benefits, 2 groups were compared: group A (27 patients: 26 men, 1 woman) with an initial success; 1 patient was lost to follow-up (3.7%); group B (33 patients, 31 men, 2 women) with an initial failure; no patients were lost to follow-up and 1 patient died after secondary coronary artery surgery. After a follow-up of at least 1 year, the patients assessed their health and compared it with their pre-angioplasty state with the aid of a questionnaire. Age, initial left ventricular ejection fraction, the percentage of multiple vessel disease and the number of patients at work before angioplasty were the same in the two groups. After a mean follow-up period of two and a half years, 7.6% of patients had undergone coronary bypass surgery in group A compared with 18.2% in group B (NS); 69% of patients in group A claimed to be improved compared with only 24% in group B (p = 0.07); a bi or triple anti-anginal therapy was used in 58% of patients in group B and 42% of patients in group A (NS); however, 59% of patients returned to work in group B compared with 44% in group A (NS). Initial successful recanalisation of chronic right coronary occlusion improves the quality of life at long-term but does not increase the chances of returning to work.(ABSTRACT TRUNCATED AT 250 WORDS)

[Influence of the severity of coronary stenosis on the course of left ventricular function in case of subsequent coronary occlusion. Longitudinal coronarographic study]. / Influence de la sévérité d'une sténose coronaire sur l'évolution de la fonction ventriculaire gauche en cas d'occlusion coronaire ultérieure. Etude coronarographique longitudinale.

In order to evaluate the consequences concerning left ventricular function of the spontaneous occlusion of coronary stenoses, the authors studied the clinical and angiographic characteristics of 30 consecutive patients (25 men, 5 women, mean age: 54) undergoing successive coronary arteriograms showing progression to complete occlusion of the anterior interventricular or right coronary between the two investigations. Two groups of patients were identified: Group I (n = 19) with occlusion of a previously moderate (< or = 50%) stenosis; Group II (n = 11) with occlusion of an initially tight stenosis (> 50%). At the time of the first angiogram, left ventricular ejection fraction (LVEF) was 60 +/- 13% in Group I and 58 +/- 9% in Group II (NS). Times between the two investigations were similar in the two groups (58 +/- 43 months and 54 +/- 57 months, NS). Between the two coronary arteriograms, 7 patients of Group I sustained an infarction as against 3 in Group II (NS). Impairment of LVEF developed in 16 patients of Group I (84%) as against 5 of Group II (45%) (p < 0.005). Global LVEF varied on average by -10.4 +/- 13.3% (p < 0.005) in Group I and -0.1 +/- 9.5% (NS) in Group II. This difference in variation in LVEF between the two groups was significant (p < 0.04). Spontaneous occlusion of moderate coronary stenoses results in certain cases in greater impairment of left ventricular function than the occlusion of tight stenoses. The development of a collateral circulation probably plays a protective role in the second group.

Effect on early acute occlusion rate of adjunctive antithrombotic treatment with intravenously administered dipyridamole during percutaneous transluminal coronary angioplasty.

This study compared the acute occlusion and complication rates within 24 hours of coronary angioplasty in three groups of patients. In group 1, 178 procedures were performed by one operator who administered 30 mg of dipyridamole intravenously over 1 hour, starting immediately before the procedure; in group 2, 200 procedures were performed by the same operator before he administered dipyridamole; and in group 3, 599 procedures were performed during the same time period in the same catheterization laboratory by two other operators who did not administer dipyridamole. All patients received an intravenous bolus of heparin and aspirin. Baseline variables were similar in the three groups. The acute closure rate was 2.8% in group 1, 7.5% in group 2, and 5.2% in group 3 (p < 0.05 between groups 1 and 2); acute thrombosis was observed in 0.6%, 3.5%, and 3% of patients, respectively, in the three groups (p < 0.05 between group 1 and both groups 2 and 3), and acute dissection was noted in 2.2%, 4%, and 2% of patients, respectively (p = not significant). The cumulative rate of acute complications (death, acute myocardial infarction, or emergency coronary bypass surgery) was lower in group 1 (1.7%) than in group 2 (5.5%, p < 0.05) and group 3 (3.5%, p = not significant). Therefore in this retrospective study, adjunctive antithrombotic treatment with intravenously administered dipyridamole resulted in lower acute thrombosis and complication rates during the 24-hour period after the procedure than when heparin and aspirin therapy were used alone.

Long-term follow-up after coronary angioplasty in patients younger than 40 years of age.

Over an 11-year period, the initial and late outcomes of percutaneous transluminal coronary angioplasty (PTCA) were studied in 140 consecutive patients younger than 40 years of age (mean, 34 +/- 3 years; range, 23 to 39 years; 132 men). Before the procedure, 28% of the patients had unstable angina, and 44% had a history of prior myocardial infarction. Mean left ventricular ejection fraction was 64% +/- 10%, and 75% of the patients had one-vessel disease. Primary success was 86% (77% for the first 70 patients vs 93% for the last 70, p < 0.02). Complications were nine periprocedural myocardial infarctions, eight emergency coronary surgical procedures, and no deaths. During follow-up (mean, 6 +/- 3 years; range, 1 to 12 years), 39 (28%) of the 104 patients who had repeat coronary angiography had angiographic restenosis (all < 6 months after PTCA). Late events were 13 elective coronary surgical procedures (11 for restenosis, one for failed PTCA, and one for progression of coronary artery disease), 13 PTCAs on a new site, five deaths, and four nonfatal myocardial infarctions. Ten-year survival was 96% +/- 1%, and 10-year event-free survival (without myocardial infarction, elective coronary surgery, or repeat PTCA) was 58% +/- 6%. Among survivors, 88% were free of angina, and 93% had returned to work. In patients younger than 40 years of age, PTCA yields excellent long-term survival, provided that the eventuality of repeat procedures during the first months is accepted. In addition, PTCA for progression on a new site is not unusual after several years.

Microparticle-enhanced nephelometric immunoassay of anti-thyroid peroxidase autoantibodies in thyroid disorders.

Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.