PARADOXICAL EXPANSION OF INTRACRANIAL TUBERCULOMAS DURING CHEMOTHERAPY

181

of infants in whom ventriculomegaly was identified at a late stage. In those studies that have included neonates with ventriculomegaly identified shortly after birth, large head circumference has been used as an inclusion criterion,8.16 even though it is well known that ventriculomegaly often precedes unusually large head size. 1.19 Therefore our study of fetuses with ventriculomegaly, with or without macrocephaly, shows less

comprised

selection bias. There were no spontaneous antepartum deaths: Unlike other fetal anomalies such as cystic hygroma,22 fetal ventriculomegaly does not appear to be associated with intrauterine death. 8 of the 23 deaths (35%) were associated with intrapartum cephalocentesis and another 8 (35%) resulted from severe congenital abnormalities. These 2 important causes of perinatal death have not been sufficiently recognised in previous studies of the outcome of fetal

ventriculomegaly. 2-18 There was abnormalities

only 1 survivor who had no associated (case 1), and the findings of normal Bayley scores and neurological features in this infant were not surprising. Normal, even superior, intelligence has been reported in cases of isolated severe neonatal

PARADOXICAL EXPANSION OF INTRACRANIAL TUBERCULOMAS DURING CHEMOTHERAPY W. A. HENDRICKSE† S. T. CHAMBERS* PETER RUDGE‡ C. RECORD* HILLAS SMITH* *Lister Unit and Department of Radiology, Northwick Park Hospital, Harrow, Middlesex; † Hospital for Sick Children, Great Ormond Street, London; and ‡National Hospital for Nervous Diseases, Queen Square, London

Summary

4 patients with tuberculosis, 3 of whom had tuberculous meningitis, were noted to have

tuberculomas on computed tomographic scanning. During antituberculous chemotherapy the intracranial lesions increased in size in all 4 patients at a time when the clinical state and cerebrospinal-fluid abnormalities were improving; in 2 of the patients the regional lymph nodes also enlarged greatly. Though the expansion of the cerebral lesions caused anxiety and led to some changes in chemotherapy, the lesions eventually diminished in size.

ventriculomegaly. 5, 7,10,11,15,16 There

Introduction

survivors with

ventriculomegaly and associated meningomyelocele (cases 2-7). Of these, 4 had normal intelligence, 1 had borderline intelligence, and 1 was retarded. Meningitis with klebsiella and pneumococcus might have caused the retardation (case 7). These findings are consistent with results of larger neonatal studies of infants with ventriculomegaly and meningomyelocele. 7,10-12,15,166 Of the 7 infants with ventriculomegaly and other associated abnormalities (cases 8-14), only case 8 had a normal were

6

In 4, cases 9, 10, 12, and 14, the poor outcome resulted from the severity of the associated anomalies. However, the poor outcomes of cases 11 and 13 were not clearly related to digital abnormalities, ventricular septal defect, or postnatal morbidity. In these latter 2 cases, the minor abnormalities may have been markers of a brain dysgenesis more severe than isolated ventriculomegaly. The presence of microcephaly in both of these infants supports this suggestion. Further clinical investigation concerning the association of fetal ventriculomegaly with developmental outcome is required. With larger study populations it would be possible to examine the effects on outcome of such variables as the presence and nature of specific associated anomalies, gestational age at onset of ventriculomegaly, the severity and rate of increase of the ventriculomegaly, and the severity of prenatal macrocephaly. Such research would be most helpful to both physicians and parents when considering antepartum and intrapartum obstetric options. outcome.

probably

B’e thank Maurice J. Mahoney, MD, of the Department of Human Genetics of Yale University, for his support, encouragement, and careful review of the manuscript.

Correspondence should be addressed to F. A. C., Department of Obstetrics,

Gynecology and Reproductive Science, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA, from whom further antenatal and clinical data about the 14

survivors

may be obtained.

REFERENCES

FA, Berkowitz RL, Romero R, et al. The diagnosis offetal hydrocephalus Am J Obstet Gynecol 1983; 147: 703-16. 2 Laurence KM, Coates S. The natural history of hydrocephalus Detailed analysis of 182 unoperated cases. Arch Dis Child 1962; 37: 345-62. 1 Chervenak

References conlllllled at foot of next colzimn

recognised as part of the classical in the brain of patients with found pathological changes tuberculous meningitis. Computed tomography (CT) brain scans have shown the presence of abnormalities in such patients which are ascribed to the presence of tuberculomas.’-6 These lesions have been noted to develop during the course of successful treatment of tuberculous TUBERCULOMAS

are

F. A. CHERVENAK AND OTHERS:

REFERENCES-continued

EL, Shurtlef DB Five-year comparative study of hydrocephalus in children with and without operation (113 cases). J Neurosurg 1963; 20: 1064-79. 4. Yashon D, Jane JA, Sugar O. The course of severe untreated infantile hydrocephalus. Prognostic significance of the cerebral mantle. J Neurosurg 1965, 23: 509-16. 5. Lorber L The results of early treatment of extreme hydrocephalus. Devel Med Child Neurol 1968; suppl 16: 21-29. 6. Lorber J, Zachary RB. Primary congenital hydrocephalus. Long-term results of controlled therapeutic trial Arch Dis Child 1968; 43: 516-27 7. Shurtleff DB, Foltz EL, Loeser JD. Hydrocephalus. A definition of its progression and relationship to intellectual function, diagnosis, and complications Am J Dis Child 1973; 125: 688-93. 8. Mealey J, Gilmor RL, Bubb MP. The prognosis of hydrocephalus overt at birth. J Neurosurg 1973; 39: 348-55. 9. Young HF, Nulsen FE, Weiss MH, Thomas P. The relationship of intelligence and cerebral mantle in treated infantile hydrocephalus. Pediatrics 1973; 52: 38-44. 10. Raimondi AJ, Soare P Intellectual development in shunted hydrocephalic children. Am J Dis Child 1974; 127: 664-71. 11. Shurtleff DB, Kronmal R, Foltz EL. Follow-up comparison of hydrocephalus with and without myelomemngocele. Neurosurgery 1975; 42: 61-68. 12. Soare PL, Raimondi AJ Intellectual and perceptual-motor characteristics of treated myelomeningocele children. Am J Dis Child 1977; 131: 199-204. 13. Keucher TR, Mealey J. Long-term results after ventriculoatrial and ventriculoperitoneal shunting for infantile hydrocephalus. J Neurosurg 1979; 50: 179-86 14. Sutton LN, Bruce DA, Schut L. Hydrencephaly versus maximal hydrocephalus: An important clinical distinction. Neuro surgery 1980; 6: 35-38 15. Dennis M, Fitz CR, Netley CT, et al. The intelligence ofhydrocephalic children. Arch Neurol 1981; 38: 607-15. 16. McCullough DC, Balzer-Martin LA. Current prognosis in overt neonatal hydrocephalus J Neurosurg 1982; 57: 378-83. 17. Jansen J, Gloerfelt-Tarp B, Pedersen H, Zilstorff K Prognosis in infantile hydrocephalus. Follow-up in adult patients, born 1946-1955. Acta Neurol Scand 1982; 65: 81-93. 18. Liechty EA, Gilmore RL, Bryson CQ, Bull MJ Outcome of high-risk neonates with ventriculomegaly. Develop Med Child Neurol 1983; 25: 162-68 19. Jeanty P, Dramaix-Wilmet M, Delbeke D, et al. Ultrasonic evaluation of fetal ventricular growth Neuroradiology 1981; 21: 127-32. 20. Chervenak FA, Jeanty P, Cantraine F, et al. The diagnosis of fetal microcephaly. Am J Obstet Gynecol (in press) 21. Usher R, McLean F Intrauterine growth of live-born Caucasian infants at sea level: Standards obtained from measurements in 7 dimensions of infants born between 25 and 44 weeks of gestation. J Pediatr 1969, 74: 901-10. 22. Chervenak FA, Isaacson G, Blakemore KJ et al. Fetal cystic hygroma. Cause and natural history. N Engl J Med 1983; 309: 822-25 3. Foltz

182

Fig

1-Case 1: CT brain

scan.

A, left temporal lobe tuberculoma is surrounded considerably smaller.

by oedema (arrow). B,

7 months later the tuberculoma has increased

meningitis.7.8

We describe 4 patients with tuberculosis (3 of whom had tuberculous meningitis) in whom intracranial tuberculomas developed or increased in size during chemotherapy as judged by cranial CT scanning, while the other manifestations of their disease were responding satisfactorily. In 2 of the patients enlargement of the regional lymph nodes was noted about the time of radiological expansion of the cerebral lesions.

Case-reports Case1 A 25-year-old Indian woman, living in England, returned to visit her family in Bombay in December, 1982. She became ill with fever, severe headache, and vomiting. The symptoms persisted, and in early January, 1983, she was admitted to hospital, where she was noted to be drowsy, with neck stiffness and papilloedema. The cerebrospmal fluid (CSF) was under increased pressure and contained 330x 106 leucocytes/litre (70% lymphocytes), 1-08 g/litre protein, and reduced glucose concentration. Gram-staining of CSF smears was unrevealing, and specific stains for acid-fast bacilli and for cryptococci were negative. She was treated with penicillin, streptomycin, isoniazid, and rifampicin, and 2 weeks later ethambutol was added to this regimen. There was a good clinical response and on Feb 9 she was discharged from hospital afebrile with occasional headaches. Streptomycin was discontinued. She remained well until March 12, when she had her first complex partial seizure; while reading aloud she heard music in the right ear and began to speak using the wrong words, but was able to understand people who spoke to her. This episode lasted 15-20 min. She recovered completely. On admission to hospital an

A.

an area

CT brain

of infarction

scan. is seen in

the left middle cerebral

arterv

territory

a

1 year later the tuberculoma is

left

temporal slow-wave

abnormality and a CT brain scan demonstrated a left temporal mass (fig 1A). At this time right cervical lymphadenopathy was first noted. After a second fit phenobarbitone and phenytoin were introduced and dexamethasone was given. She returned to England and was admitted to the National Hospital for Nervous Diseases on March 30.

only abnormality noted on clinical examination was a large of lymph nodes in the right supraclavicular fossa. A repeat CT brain scan showed that the left temporal space-occupying lesion was essentially unchanged. The CSF contained 90x 106 mononuclear cells/litre, protein 0-9 g/l, and sugar 2 - 4 mmol/1 (blood glucose 4 - 6 mmol/1). Organisms were not seen on smear, and the diagnosis was considered to be resolving tuberculous meningitis with a left temporal lobe tuberculoma and tuberculous lymphadenopathy. The rifampicin and isoniazid were increased, and the dexamethasone was gradually withdrawn. She remained well until a further fit on April 19, when her supraclavicular lymph nodes were noted to be grossly enlarged. The repeat CT brain scan at this time suggested that the left temporal lobe lesion might have become larger. The CSF changes were similar to those previously noted. Pyrazinamide was added to her antituberculous therapy. At this stage she was transferred to the Lister unit at Northwick Park Hospital. The large fluctuant supraclavicular mass was aspirated, and thick pus was obtained. Acid-fast bacilli were present, but culture for mycobacteria was always negative. The mass did not resolve, and on May 17 a large amount of necrotic material was removed surgically. CT brain scans on May 4 and June 10 demonstrated that the left temporal mass was surrounded by an increasing area of oedema, and The

mass

a

further

scan

in October demonstrated that the lesion itself was well

probably larger (fig 1B). Clinically, however, the patient was

CT brain

scan.

tuberculoma is seen in the mesencehalon (curved arrow) and a second in the left caudate nucleus (straight arrow). B, 2’/2 months later the mesencephalic tuberculoma has enlarged (curved arrow) The tuberculoma in the caudate nucleus is smaller (straight arrow).

A,

(arrows). the ventricles are moderately dilated. B,5 months later a tuberculous abscess has developed tn the right frontal lobe (large arrow).

C,

electroencephalogram (EEG) showed

Fig 3-Case 4: Fig 2-Case 2:

in size.

one

183 apart from occasional fits, and she continues to have regular antituberculous therapy. Brain scan on March 10, 1984, showed that the tuberculoma was smaller and the oedema had resolved (fig

1 C). Case 2 A 9-year-old Indian girl was found to have tuberculous meningitis with typical CSF abnormalities including the presence of acid-fast bacilli. Mycobacterium tuberculosis was eventually cultured. She was transferred to the Hospital for Sick Children, Great Ormond Street, because of progressive drowsiness and papilloedema. A CT brain scan showed contrast enhancement of the basal meninges, infarction in the left middle cerebral territory, and moderate hydrocephalus (fig 2A). She gradually improved with antituberculous therapy, but 5 months after the onset of her illness she became obese with compulsive eating and complained of headaches. Repeat CT brain scanning demonstrated a large ring enhancing lesion in the inferior part of the right frontal lobe (fig 2B) from which 9 ml of pus containing numerous acid-fast bacilli was aspirated. Culture was sterile. Repeat CT scans showed that the abscess was slightly smaller, but a second small abscess was noted in the frontal lobe. 10 months after the start of treatment a rapidly expanding supraclavicular node appeared and was excised. No acidfast bacilli were seen, and culture was sterile.

Case 3 A

34-year-old Kenyan Asian was admitted to the Lister Unit in August, 1979, with a week’s history suggestive of meningitis. The CSF changes were compatible with tuberculous infection, and acidfast bacilli

were

seen.

M tuberculosis sensitive

to

standard

chemotherapy was subsequently grown. Chest X-ray showed miliary mottling. Standard chemotherapy was started. A CT brain scan 14 days after admission, carried out because of disorientation and hallucination, showed no hydrocephalus, but there was a 1 cm area of increased attenuation in the left frontal region, and a similar lesion was present in the right posterior frontal region. He improved but continued to complain of headache, was withdrawn, and had frequent auditory hallucinations. A psychiatrist diagnosed schizophenia. He was discharged from hospital in October, 1979, but was readmitted to the Lister unit in March, 1980, after a major convulsion. CT scan showed that the previously noted lesions had increased in size and were surrounded by a zone of oedema. The dosage of antituberculous drugs was increased. The fits were controlled with anticonvulsants; and the patient’s general condition improved, with reduction in the frequency of hallucinatory episodes. Despite the clinical improvement, a CT brain scan in May, 1980, showed oedema in the right frontoparietal region, and contrast-enhancing nodules were again identified. In February, 1981, he returned to work. A CT brain scan in February, 1984, was normal.

Case4 A 24-year-old Indian woman presented with a 4-month history of intermittent pyrexia and a 3-week history of intermittent diplopia, ptosis, and weight-loss. Examination showed bilateral ptosis. Horizontal movements of the right eye were confined to a few i degrees of abduction; vertical movements were full. Movements of ! the left eye were normal. There was primary position up-beating nystagmus. The remainder of the neurological examination was normal. On general examination there were signs of a left pleural effusion, which was confirmed on chest X-ray. The pleural aspirate contained numerous acid-fast bacilli, and M tUberculosis was subsequently grown on culture. A pleural biopsy showed the presence of caseating granulomata. A CT brain scan demonstrated a small enhancing lesion in the mesencephalon at the intercollicular level, and a second lesion in the left caudate nucleus (fig 3A). CSF was normal. Antituberculous therapy with rifampicin, ethambutol, and pyrazinamide was started; the organisms were subsequently shown to be sensitive to all three drugs. 6 days later the patient became drowsy and complained of headaches. The CT brain scan was unchanged but her condition improved on dexamethasone. The fever rapidly settled over 4 ,

and the pleural effusion completely resolved; dexamethasone was stopped and the antituberculous therapy continued. She remained systemically well, although ocular symptoms and signs persisted. A repeat CT brain scan 10 weeks after therapy was instituted demonstrated an increase in size in the mesencephalic lesion, which now appeared to have an enhancing rim. The caudate nuclear lesion was if anything smaller (fig 3B). After 9 months of antituberculous therapy she began to complain of headache and blurred vision. On admission 3 months later she had a complex bilateral ophthalmoplegia with some down-beat nystagmus. The remainder of the examination was normal, and-the chest X-ray was clear. A CT brain scan showed hydrocephalus, and the mesencephalic enhancing lesion was slightly less prominent than previously. The left caudate nucleus lesion was no longer present.

weeks,

Discussion Our 4 patients showed progressive enlargement of intracranial tuberculomata detected on CT brain scanning at a time when the other systemic features of the illness (except lymphadenopathy in cases 1 and 2) were improving, and the CSF abnormalities in the 3 patients with tuberculous

meningitis were resolving. The time course of these lesions is complex. They were identified on CT scans done soon after admission and were clincally silent; suspicion that they were causing progressive clinical disease did not arise until 2-7 months after starting treatment. During this period the lesions increased in size and in most cases were surrounded by oedema and on one occasion showed ring enhancement with abscess formation. In 1 patient (case 2) a secondary lesion became apparent 7 months after treatment began. Resolution of these lesions may take up to a year. We report these observations so that others who encounter similar manifestations, especially at a time when the diagnosis of tuberculous meningitis is suspected rather than proved, may be aware that anxiety about alternative space-occupying lesions is likely to be misplaced, and that the progressive CT brain scan abnormalities do not in themselves demand alteration of antituberculous chemotherapy. We have no idea how common these findings are, but they are unlikely to be rare. Like others we have observed some patients with tuberculous meningitis who did not produce CT abnormalities and some patients with tuberculomas which resolved during therapy. The explanation of the paradoxical expansion of the lesions is obscure. There was good evidence in our patients that the chemotherapy was appropriate; furthermore, the pus obtained from the intracranial lesion in case 2 and from lymph nodes in cases 1 and 2, although containing acid-fast bacilli (case 1), was sterile. Possibly the increase in size of the intracranial tuberculomas has an immunological basis. These changes seem similar to the enlargement of regional lymph nodes seen in patients being adequately treated for glandular

generally

tuberculosis.9 REFERENCES

RC, Shawdon HH. Five cases of intracranial tuberculomata followed by serial computerised tomography J Neurol Neurosurg Psychiatry 1979; 42: 373-79. 2. Casselman ES, Hasso AN, Ashwal S, Schneider S. Computed tomography of tuberculous meningitis in infants and children. J Comp Assist Tomography 1980; 4: 1. Peatfield

211-16. 3 Nai-Shin Chu Tuberculous meningitis: Computerized tomographic manifestations. Arch Neurol 1980; 37: 458-60. 4. Grabman JP, Kompare EA Computerized tomography in the management of cerebral tuberculomas. West J Med 1980; 133: 345-46 5. Bhargava S, Gupta AK, Tandon PN Tuberculous meningitis—a CT study. Br J Radiol 1982, 55: 189-96.

ReJerences coml1lllcd overleaf

184

AFFINITY OF ANTIBODY RESPONSES IN MAN TO HEPATITIS B VACCINE DETERMINED WITH SYNTHETIC PEPTIDES S. E. BROWN A. J. ZUCKERMAN

C. R. HOWARD M. W. STEWARD

Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, London WC1 7HT The affinity and level of antibody to hepatitis B surface antigen (anti-HBs) in recipients of a plasma-derived hepatitis B vaccine were determined with three different antigens. The first two antigens were prepared by chemical synthesis, to represent

Summary

linear or cyclical forms of aminoacid sequences 139 to 147 of the major hepatitis B surface antigen (HBsAg) polypeptide. The binding of antibodies to these synthetic peptides was compared with that to a third antigen, prepared by solubilisation of the naturally occurring HBsAg, the basic component of the currently licensed hepatitis B vaccine in the United Kingdom. Antibody levels, expressed as total antibody combining sites (Abt) in fixed volumes of immune sera, increased throughout the course of immunisation and correlated with the development of antibody as measured by a commercially available radioimmunoassay. Abt values were similar for both forms of the synthetic peptide, although higher affinity values were found with the cyclical structure, which illustrates the importance of protein conformation in antibody responses to HBsAg. Antibody affinity for the three antigens increased progressively throughout the immunisation schedule but the pattern of affinity maturation varied according to the peptide used as an antigen probe and between subjects. Most subjects showed a significant rise in antibody affinity after the third (booster) dose of vaccine given at six months. The use of synthetic peptides allowed a quantitative and qualitative assessment of antibody responses to hepatitis B vaccine and confirmed that selected peptides corresponding to relevant HBsAg epitopes may be useful as alternative hepatitis B vaccines.

Introduction CURRENT hepatitis B vaccines licensed for human use consist of highly purified HBsAg particles obtained from the plasma of persistently infected individuals.’ These preparations are both safe and effective in various population groups such as health care personnel and patients at high risk of acquiring hepatitis B.2Although seroconversion rates may reach 95°7o among healthy persons, antibody production assessed by radioimmunoassay may be poor in individuals over the age of 40 or in patients on maintenance haemodialysis (ref 3 and Hilleman MR and McLean A, personal communication). Given the source of current hepatitis B vaccines and the purification procedures and vigorous safety testing required to ensure freedom from all contaminating transmissible agents, there is an urgent need to develop safe and effective vaccines from alternative sources of HBsAg. In addition, the apparent variability in immune responses to the current vaccines, together with the lack of information concerning the quality of specific antibody in S. T. CHAMBERS AND OTHERS:

REFERENCES—continued

AJ, MacLeod AF, Marshall J. Cerebral tuberculomas developing during treatment of tuberculous meningitis. Lancet 1980; i: 1208-11. Lebas J, Malkin JE, Coquin Y, Modai J. Cerebral tuberculomas developing during

6. Lees 7.

of tuberculous meningitis. Lancet 1980; ii 84. LA, Anderson M Intracranial tuberculomas: Correlation of computerized tomography with clinico-pathological findings. Quart J Med 1982; 202: 104-14. Campbell IA, Dyson AJ Lymph node tuberculosis: A comparison of various methods treatment

8. Loizou 9.

of treatment. Tuberculosis 1977; 58: 171-79.

chronically infected individuals, make the development of qualitative tests with specific antigen probes highly desirable. Antibody quality can be expressed in terms of the binding affinity of the antibody for the homologous antigen, which is an important factor in determining the biological properties of antisera. 4,5 Purified HBsAg particles consist of two major polypeptides of molecular weights 23 000 (p23) and 28 000 (gp28).6 Both contain the group determinant a present on all particles and known to correlate with the induction of protecting antibody responses.7 The chemical synthesis of small peptides representing specific regions of the HBsAg polypeptides is now feasible, with the deduction of total ;‘ aminoacid sequences by direct sequencing of the hepatitis B virus genome. Such peptides resembling selected regions of the HBsAg molecule may be coupled to carrier molecules and have been used as immunogens in laboratory animals.8-" Antisera to these peptides cross-react with the native HBsAg particle, which indicates the potential of these peptides as alternative hepatitis B vaccines. Antibodies to one such aminoacid sequence has been directly demonstrated in human sera containing anti-HBs.12 A nonapeptide representing aminoacids 139 to 147 of the 226 aminoacid HBsAg molecule bound antibodies present in a variety of sera which included a national standard preparation of human immunoglobulin used prophylactically in cases of accidental exposure to hepatitis B. To extend these observations, the ability of antibodies found in individuals receiving the currently licensed hepatitis B 22 nm particle vaccine (’Hep-B-Vax’, Merck, Sharp and Dohme) to bind to two conformationally distinct forms of the synthetic peptide was compared with their ability to bind to the native HBsAg molecule containing all possible antigenic sites. In addition, the affinity of specific antibody produced by the vaccine was measured by taking advantage of the specificity of the synthetic peptide as a probe for antibody binding. This study has important implications both for the assessment of vaccine efficacy and for providing further evidence that, with identification of the relevant immunogen, hepatitis B vaccines can be chemically synthesised.

Subjects

and Methods

Serum samples were obtained from 19 healthy individuals at various times (table I) during immunisation with a licensed plasmaderived hepatitis B vaccine (’Hep-B-Vax’, Merck, Sharp and Dohme). All were laboratory workers working on various aspects of hepatitis B. Serum samples were obtained on each of five occasions from 8 subjects, and at least one post-vaccination sample was available from all individuals at the time of seroconversion. Each time, the sera were first tested for antibody to HBsAg by solid-phase radioimmunoassay (’Ausab’, Abbott Laboratories, North Chicago) and only those sera found positive were evaluated further for anti. body binding and affinity. The two synthetic peptides represented aminoacids 139 to 147 and were prepared either as linear or cyclical peptides. Each was prepared by solid-phase chemistry by Cambridge Research Biochemicals Ltd, Harston, Cambridge, and contained an additional tyrosine residue at the carboxyl terminal to facilitate radiolabelling. 12 The native HBsAg particles containing the 23 000 mol wt (p23) polypeptide and its glycosylated form (gp28) were prepared as a soluble complex following dissociation in 2% ’Triton

X-100’ (Sigma, London).13

Affinities of antibodies to HBsAg in vaccinees were determined with the three different 125l-labelled antigens as described Briefly, 10 <1 of serum was incubated at 4°C for 1 h with a range of antigen concentrations (6-700 pmol) in a final volume of 50 pl. At equilibrium, the free and antibody-bound antigen concentrations were determined after precipitation with

previously. 12,14-16